Immune-derived moieties reactive against lysophosphatidic acid

ABSTRACT

Compositions and methods for producing monoclonal antibodies and their derivatives reactive against bioactive lipid targets are described. These compositions include derivatized lipids, each of which comprises a bioactive lipid that having a polar head group and at least one hydrocarbon chain (e.g., a lysolipid such as lysophosphatidic acid or sphingosine-1-phosphate) in which a carbon atom has been derivatized with a pendant reactive group; immunogens made by linking a derivatized lipid to a carrier moiety (e.g., a carrier protein, polyethylene glycol, colloidal gold, alginate, or a silicone bead); monoclonal antibodies and derivatives produced by immunizing an animal with such an immunogen; and therapeutic and diagnostic compositions containing such antibodies and antibody derivatives. Methods for making such derivatized lipids, immunogens, and monoclonal antibodies and derivatives, methods for detecting such antibodies once generated, and therapeutic and diagnostic methods for using such antibodies and derivatives, are also described.

RELATED APPLICATIONS

This patent application claims priority to U.S. provisional patentapplication Ser. No. 60/810,185, filed 31 May 2006, U.S. provisionalpatent application Ser. No. 60/835,569, filed 4 Aug. 2006, and U.S.provisional patent application Ser. No. 60/923,644, filed 16 Apr. 2007.These applications are hereby incorporated by reference in theirentirety for any and all purposes.

GOVERNMENT FUNDING

This invention was funded at least in part by funds supplied by the U.S.government pursuant to grant application NCI 2R44CA110298-2. As aresult, the U.S. government may have certain rights in the inventionsdescribed herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Aug. 4, 2009, is namedLPT3100UT4.txt, and is 53,940 bytes in size.

TECHNICAL FIELD

The present invention relates to monoclonal antibodies, and methods forgenerating antibodies against immunogens that comprise a bioactive lipidmolecule that plays a role in human and/or animal disease as a signalingmolecule. One particular class of signaling bioactive lipids that can beaddressed in accordance with the invention is lysolipids. Particularlypreferred signaling lysolipids are sphingosine-1-phosphate (S1P) and thevarious lysophosphatidic acids (LPAs). The antibodies of the inventioncan be further modified to make them suitable for use in a particularanimal species, including humans, without eliciting a neutralizingimmune response. Such antibodies, and derivatives and variants thereof,can be used in the treatment and/or prevention of various diseases ordisorders through the delivery of pharmaceutical compositions thatcontain such antibodies, alone or in combination with other therapeuticagents and/or treatments. In addition, the antibodies can be also beused to detect bioactive signaling lipids in biologic samples, therebyproviding useful information for many purposes including, but notlimited to, the diagnosis and/or prognosis of disease and the discoveryand development of new treatment modalities that modify the productionand or actions of the particular targeted lipid. The diseases orconditions to be affected by the compositions of the invention include,but are not limited to, diseases that have hyperproliferation,angiogenesis, inflammation, fibrosis, and/or apoptosis as part of theirunderlying pathology.

BACKGROUND OF THE INVENTION

1. Introduction

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that anysuch information is prior art, or relevant, to the presently claimedinventions, or that any publication specifically or implicitlyreferenced is prior art or even particularly relevant to the presentlyclaimed invention.

2. Background

A. Bioactive Signaling Lipids

Lipids and their derivatives are now recognized as important targets formedical research, not as just simple structural elements in cellmembranes, solubilizing agents, feedstock for vitamins or hormones or asa source of energy for β-oxidation, glycolysis or other metabolicprocesses. In particular, certain bioactive lipids function as signalingmediators important in animal and human disease. Although most of thelipids of the plasma membrane play an exclusively structural role, asmall proportion of them are involved in relaying extracellular stimuliinto cells. “Lipid signaling” refers to any of a number of cellularsignal transduction pathways that use bioactive lipids as first orsecond messengers, including direct interaction of a lipid signalingmolecule with its own specific receptor. Lipid signaling pathways areactivated by a variety of extracellular stimuli, ranging from growthfactors to inflammatory cytokines, and regulate cell fate decisions suchas apoptosis, differentiation and proliferation. Research into bioactivelipid signaling is an area of intense scientific investigation as moreand more bioactive lipids are identified and their actionscharacterized.

Examples of bioactive lipids include the eicosanoids derived fromarachidonic acid (including the eicosanoid metabolites such as theHETEs, cannabinoids, leukotrienes, prostaglandins, lipoxins,epoxyeicosatrienoic acids, and isoeicosanoids), non-eicosanoidcannabinoid mediators, phospholipids and their derivatives such asphosphatidic acid (PA) and phosphatidylglycerol (PG) and cardiolipins aswell as lysophospholipids such as lysophosphatidyl choline (LPC) andvarious lysophosphatidic acids (LPA). Bioactive signaling lipidmediators also include the sphingolipids such as ceramide,ceramide-1-phosphate, sphingosine, sphinganine,sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P).Sphingolipids and their derivatives represent a group of extracellularand intracellular signaling molecules with pleiotropic effects onimportant cellular processes. Other examples of bioactive signalinglipids include phosphatidylinositol (PI), phosphatidylethanolamine(PEA), diacylglyceride (DG), sulfatides, gangliosides, and cerebrosides.

As expected, biological lipids (i.e., lipids that occur in nature,particularly in living organisms) are typically non-immunogenic or veryweakly immunogenic. As such, lipids have traditionally been consideredto be poor targets for antibody-based therapeutic anddiagnostic/prognostic approaches. The literature contains a report of amonoclonal antibody that targets a derivatized form ofphosphatidylserine (PS) conjugated to a carrier protein.Phosphatidylserine is a plasma membrane aminophospholipid. Loss ofmembrane lipid sidedness, in particular the emergence ofphosphatidylserine at the cell surface, results in the expression ofaltered surface properties that modulates cell function and influencesthe cells interaction with its environment [Zwaal and Schroit, (1997)Blood, 89:1121-1132]. For example, PS redistributes from the cellmembrane's inner leaflet (its normal location) to the outer leafletduring apoptosis.

Diaz, Balasubramanian and Schroit [Bioconj. Chem. (1998) 9:250-254]disclose production of lipid antigens that elicit specific immuneresponses against PS. The covalent coupling of PS to a protein carrier(BSA) via the lipid's fatty acyl side chain preserves the PS head groupintact as an epitope. Schroit (U.S. Pat. No. 6,300,308, U.S. Pat. No.6,806,354) discloses antibodies that specifically bind tophosphatidylserine (PS) or a phosphatidylcholine (PC)/polypeptide or aPS/polypeptide conjugate, that are made by administering aPS/polypeptide conjugate or a PC/polypeptide conjugate to an animal.Methods for detecting PS, a PC/polypeptide or a PS/polypeptide conjugateare also disclosed. Methods for making an antibody that specificallybinds to PS by administering to an animal a pharmaceutical compositioncomprising a PS/polypeptide conjugate composition are also disclosed, asare methods for treating cancer in the animal to which the conjugate isadministered, i.e., as a cancer vaccine. Also disclosed is induction ofautoimmunity for the therapy of cancer by immunization of animals withβ2-glycoprotein I/lipid complexes (i.e., non-covalently associated lipidand glycoprotein). The authors assert that several autoimmune responsesare directed against β2-glycoprotein I/lipid complexes (citingSchousboe, (1979) Biochim. Biophys. Acta, 579:396-408), and thus thegeneration of an anti-complex response may represent substantialbreakthroughs in the treatment of cancers.

Thorpe, Schroit et al. describe a monoclonal antibody (3G4) that bindsanionic phospholipids in the presence of serum or the serum protein β2-glycoprotein I (β2-GPI). Luster et al., J. Biol. Chem. 281:29863-29871. Originally described as specifically targeting anionicphospholipids, this antibody localizes to vascular endothelial cells intumors in mice. Ran et al. (2005) Clin. Cancer Res. 11:1551-1562.Subsequently, the antibody was shown to bind to complexes of anionicphospholipids and β2-GPI on tumor vessels, so that antibody binding toPS is dependent on β2-GPI. Huang et al (2005) Cancer Res. 65:4408-4416.The antibody enhances binding of β2-GPI to endothelial cells viadimerization of β2GPI. In fact, artificial β2-GPI dimers can bind toendothelial cell membranes even in the absence of antibody. Luster etal., J. Biol. Chem. 281: 29863-29871. A humanized version of 3G4(Tarvacin, Bavituximab) is in clinical trials for treatment of cancerand viral diseases.

Thorpe et al. (WO 2004/006847) disclose antibodies, fragments orimmunoconjugates thereof that bind to PS and compete with antibody 3G4for binding to PS. Thorpe et al (U.S. Pat. No. 6,818,213, U.S. Pat. No.6,312,294 and U.S. Pat. No. 6,783,760) disclose therapeutic conjugatesthat bind to aminophospholipids and have an attached therapeutic agent.

Baldo et al. (U.S. Pat. No. 5,061,626) disclose antibodies to plateletactivating factor (PAF), PAF analogues used to generate antibodies andimmunoassays using PAF or PAF analogues. PAF is a choline plasmalogen inwhich the C-2 (sn2) position of glycerol is esterified with an acetylgroup instead of a long chain fatty acid.

Vielhaber et al. report characterization of two antibody reagentssupposedly specific for ceramide, one an IgM-enriched polyclonal mouseserum and the other an IgM monoclonal antibody. The monoclonal was foundto be specific for sphingomyelin and the antiserum was found to reactwith various ceramide species in the nanomolar range. Vielhaber, G. etal., (2001) Glycobiology 11:451-457. Also citing the deficiencies ofcommercially available antibody reagents against ceramide, Krishnamurthyet al. recently reported generation of rabbit IgG against ceramide. J.Lipid Res. (2007) 48:968-975.

B. Lysolipids

Lysolipids are low molecular weight lipids that contain a polar headgroup and a single hydrocarbon backbone, due to the absence of an acylgroup at one or both possible positions of acylation. Relative to thepolar head group at sn-3, the hydrocarbon chain can be at the sn-2and/or sn-1 position(s) (the term “lyso,” which originally related tohemolysis, has been redefined by IUPAC to refer to deacylation). See“Nomenclature of Lipids, www.chem.qmul.ac.uk/iupac/lipid/lip1n2.html.These lipids are representative of signaling, bioactive lipids, andtheir biologic and medical importance highlight what can be achieved bytargeting lipid signaling molecules for therapeutic,diagnostic/prognostic, or research purposes (Gardell, et al. (2006),Trends in Molecular Medicine, vol 12: 65-75). Two particular examples ofmedically important lysolipids are LPA (glycerol backbone) and S1P(sphingoid backbone). Other lysolipids include sphingosine,lysophosphatidylcholine (LPC), sphingosylphosphorylcholine(lysosphingomyelin), ceramide, ceramide-1-phosphate, sphinganine(dihydrosphingosine), dihydrosphingosine-1-phosphate andN-acetyl-ceramide-1-phosphate. In contrast, the plasmalogens, whichcontain an O-alkyl (—O—CH₂—) or O-alkenyl ether at the C-1 (sn1) and anacyl at C-2, are excluded from the lysolipid genus.

The structures of selected LPAs, S1P, and dihydro S1P are presentedbelow.

LPA is not a single molecular entity but a collection of endogenousstructural variants with fatty acids of varied lengths and degrees ofsaturation (Fujiwara, et al. (2005), J Biol Chem, vol. 280:35038-35050). The structural backbone of the LPAs is derived fromglycerol-based phospholipids such as phosphatidylcholine (PC) orphosphatidic acid (PA). In the case of lysosphingolipids such as S1P,the fatty acid of the ceramide backbone at sn-2 is missing. Thestructural backbone of S1P, dihydro S1P (DHS1P) andsphingosylphosphorylcholine (SPC) is based on sphingosine, which isderived from sphingomyelin.

LPA and S1P regulate various cellular signaling pathways by binding tothe same class of multiple transmembrane domain G protein-coupled (GPCR)receptors (Chun J, Rosen H (2006), Current Pharm Des, vol. 12: 161-171,and Moolenaar, W H (1999), Experimental Cell Research, vol. 253:230-238). The S1P receptors are designated as S1P₁, S1P₂, S1P₃, S1P₄ andS1P₅ (formerly EDG-1, EDG-5/AGR16, EDG-3, EDG-6 and EDG-8) and the LPAreceptors designated as LPA₁, LPA₂, LPA₃ (formerly, EDG-2, EDG-4, andEDG-7). A fourth LPA receptor of this family has been identified for LPA(LPA₄), and other putative receptors for these lysophospholipids havealso been reported.

C. Lysophosphatic Acids (LPA)

LPA have long been known as precursors of phospholipid biosynthesis inboth eukaryotic and prokaryotic cells, but LPA have emerged onlyrecently as signaling molecules that are rapidly produced and releasedby activated cells, notably platelets, to influence target cells byacting on specific cell-surface receptor (see, e.g., Moolenaar, et al.(2004), BioEssays, vol. 26: 870-881, and van Leewen et al. (2003),Biochem Soc Trans, vol 31: 1209-1212). Besides being synthesized andprocessed to more complex phospholipids in the endoplasmic reticulum,LPA can be generated through the hydrolysis of pre-existingphospholipids following cell activation; for example, the sn-2 positionis commonly missing a fatty acid residue due to deacylation, leavingonly the sn-1 hydroxyl esterified to a fatty acid. Moreover, a keyenzyme in the production of LPA, autotoxin (lysoPLD/NPP2), may be theproduct of an oncogene, as many tumor types up-regulate autotoxin(Brindley, D. (2004), J Cell Biochem, vol. 92: 900-12). Theconcentrations of LPA in human plasma and serum have been reported,including determinations made using a sensitive and specific LC/MSprocedure (Baker, et al. (2001), Anal Biochem, vol 292: 287-295). Forexample, in freshly prepared human serum allowed to sit at 25° C. forone hour, LPA concentrations have been estimated to be approximately 1.2μM, with the LPA analogs 16:0, 18:1, 18:2, and 20:4 being thepredominant species. Similarly, in freshly prepared human plasma allowedto sit at 25° C. for one hour, LPA concentrations have been estimated tobe approximately 0.7 μM, with 18:1 and 18:2 LPA being the predominantspecies.

LPA influences a wide range of biological responses, ranging frominduction of cell proliferation, stimulation of cell migration andneurite retraction, gap junction closure, and even slime mold chemotaxis(Goetzl, et al (2002), Scientific World Journal, vol. 2: 324-338). Thebody of knowledge about the biology of LPA continues to grow as more andmore cellular systems are tested for LPA responsiveness. For instance,it is now known that, in addition to stimulating cell growth andproliferation, LPA promote cellular tension and cell-surface fibronectinbinding, which are important events in wound repair and regeneration(Moolenaar, et al. (2004), BioEssays, vol. 26: 870-881). Recently,anti-apoptotic activity has also been ascribed to LPA, and it hasrecently been reported that peroxisome proliferation receptor gamma is areceptor/target for LPA (Simon, et al. (2005), J Biol Chem, vol. 280:14656-14662).

LPA has proven to be difficult targets for antibody production, althoughthere has been a report in the scientific literature of the productionof polyclonal murine antibodies against LPA (Chen et al. (2000) Med ChemLett, vol 10: 1691-3).

D. Sphingosine-1-Phosphate

S1P is a mediator of cell proliferation and protects from apoptosisthrough the activation of survival pathways (Maceyka, et al. (2002),BBA, vol. 1585: 192-201, and Spiegel, et al. (2003), Nature ReviewsMolecular Cell Biology, vol. 4: 397-407). It has been proposed that thebalance between CER/SPH levels and S1P provides a rheostat mechanismthat decides whether a cell is directed into the death pathway or isprotected from apoptosis. The key regulatory enzyme of the rheostatmechanism is sphingosine kinase (SPHK) whose role is to convert thedeath-promoting bioactive signaling lipids (CER/SPH) into thegrowth-promoting S1P. S1P has two fates: S1P can be degraded by S1Plyase, an enzyme that cleaves S1P to phosphoethanolamine andhexadecanal, or, less common, hydrolyzed by S1P phosphatase to SPH.

S1P is abundantly generated and stored in platelets, which contain highlevels of SPHK and lacks the enzymes for S1P degradation. When plateletsare activated, S1P is secreted. In addition, other cell types, forexample, mast cells, are also believed to be capable of secreting S1P.Once secreted, S1P is thought to be bound at high concentrations oncarrier proteins such as serum albumin and lipoproteins. S1P is found inhigh concentrations in plasma, with concentrations in the range of 0.5-5uM having been reported. Intracellular actions of S1P have also beensuggested (see, e.g., Spiegel S, Kolesnick R (2002), Leukemia, vol. 16:1596-602; Suomalainen, et al (2005), Am J Pathol, vol. 166: 773-81).

Widespread expression of the cell surface S1P receptors allows S1P toinfluence a diverse spectrum of cellular responses, includingproliferation, adhesion, contraction, motility, morphogenesis,differentiation, and survival. This spectrum of response appears todepend upon the overlapping or distinct expression patterns of the S1Preceptors within the cell and tissue systems. In addition, crosstalkbetween S1P and growth factor signaling pathways, includingplatelet-derived growth factor (PDGF), vascular endothelial growthfactor (VEGF), and basic fibroblastic growth factor (bFGF), haverecently been demonstrated (see, e.g., Baudhuin, et al. (2004), FASEB J,vol. 18: 341-3). The regulation of various cellular processes involvingS1P has particular impact on neuronal signaling, vascular tone, woundhealing, immune cell trafficking, reproduction, and cardiovascularfunction, among others. Alterations of endogenous levels of S1P withinthese systems can have detrimental effects, eliciting severalpathophysiologic conditions, including cancer, heart failure, andinfectious and autoimmune diseases.

A recent novel approach to treating cancer invented by Dr. Sabbadiniinvolves reducing the biologically available extracellular levels ofS1P, either alone or in combination with conventional anti-cancertreatments, including the administration of chemotherapeutic agents,such as an anthracycline. To this end, the generation of antibodiesspecific for S1P has been described. See, e.g., commonly owned U.S.patent application Ser. No. 10/820,582. Such antibodies, which canselectively adsorb S1P from serum, act as molecular sponges toneutralize extracellular S1P. See also commonly owned U.S. Pat. Nos.6,881,546 and 6,858,383 and U.S. patent application Ser. Nos.10/028,520, 10/029,372, and 11/101,976. Since S1P has also been shown tobe pro-angiogenic, an added benefit to the antibody's effectiveness isits ability to starve growing tumors of nutrients and oxygen by limitingblood supply.

What is particularly unique about the anti-S1P approach is that whilesphingolipid-based anti-cancer strategies that target key enzymes of thesphingolipid metabolic pathway, such as SPHK, have been proposed, thelipid mediator S1P itself was not previously emphasized, largely becauseof difficulties in directly mitigating this lipid target, in particularbecause of the difficulty first in raising antibodies against a lipidtarget such as S1P, and second, in detecting antibodies in fact producedagainst the S1P target. As already noted, similar difficulties existwith respect to treatments and diagnostic approaches directed at otherlipid targets. This invention provides an effective solution to both ofthese dilemmas by providing patentable methods, in particular, thegeneration of monoclonal antibodies against bioactive lipids.

3. Definitions

Before describing the instant invention in detail, several terms used inthe context of the present invention will be defined. In addition tothese terms, others are defined elsewhere in the specification, asnecessary. Unless otherwise expressly defined herein, terms of art usedin this specification will have their art-recognized meanings.

An “anti-S1P antibody” refers to any antibody or antibody-derivedmolecule that binds S1P.

A “bioactive lipid” refers to a lipid signaling molecule. Bioactivelipids are distinguished from structural lipids (e.g., membrane-boundphospholipids) in that they mediate extracellular and/or intracellularsignaling and thus are involved in controlling the function of manytypes of cells by modulating differentiation, migration, proliferation,secretion, survival, and other processes. In vivo, bioactive lipids canbe found in extracellular fluids, where they can be complexed with othermolecules, for example serum proteins such as albumin and lipoproteins,or in “free” form, i.e., not complexed with another molecule species. Asextracellular mediators, some bioactive lipids alter cell signaling byactivating membrane-bound ion channels or GPCRs or enzymes or factorsthat, in turn, activate complex signaling systems that result in changesin cell function or survival. As intracellular mediators, bioactivelipids can exert their actions by directly interacting withintracellular components such as enzymes, ion channels or structuralelements such as actin. Representative examples of bioactive lipidsinclude LPA and S1P.

Examples of bioactive lipids include sphingolipids such as ceramide,ceramide-1-phosphate, sphingosine, sphinganine,sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P).Sphingolipids and their derivatives and metabolites are characterized bya sphingoid backbone (derived from sphingomyelin). Sphingolipids andtheir derivatives and metabolites represent a group of extracellular andintracellular signaling molecules with pleiotropic effects on importantcellular processes. They include sulfatides, gangliosides andcerebrosides. Other bioactive lipids are characterized by aglycerol-based backbone; for example, lysophospholipids such aslysophosphatidyl choline (LPC) and various lysophosphatidic acids (LPA),as well as phosphatidylinositol (PI), phosphatidylethanolamine (PEA),phosphatidic acid, platelet activating factor (PAF), cardiolipin,phosphatidylglycerol (PG) and diacylglyceride (DG). Yet other bioactivelipids are derived from arachidonic acid; these include the eicosanoids(including the eicosanoid metabolites such as the HETEs, cannabinoids,leukotrienes, prostaglandins, lipoxins, epoxyeicosatrienoic acids, andisoeicosanoids), non-eicosanoid cannabinoid mediators. Other bioactivelipids, including other phospholipids and their derivatives, may also beused according to the instant invention.

In some embodiments of the invention it may be preferable to targetglycerol-based bioactive lipids (those having a glycerol-derivedbackbone, such as the LPAs) for antibody production, as opposed tosphingosine-based bioactive lipids (those having a sphingoid backbone,such as sphingosine and S1P). In other embodiments it may be desired totarget arachidonic acid-derived bioactive lipids for antibodygeneration, and in other embodiments arachidonic acid-derived andglycerol-derived bioactive lipids but not sphingoid-derived bioactivelipids are preferred. Together the arachidonic acid-derived andglycerol-derived bioactive lipids may be referred to in the context ofthis invention as “non-sphingoid bioactive lipids.”

Specifically excluded from the class of bioactive lipids according tothe invention are phosphatidylcholine and phosphatidylserine, as well astheir metabolites and derivatives that function primarily as structuralmembers of the inner and/or outer leaflet of cellular membranes.

A “biomarker” is a specific biochemical in the body which has aparticular molecular feature that makes it useful for measuring theprogress of disease or the effects of treatment.

For example, S1P is a biomarker for certain hyperproliferative and/orcardiovascular conditions.

A “carrier” refers to a moiety adapted for conjugation to a hapten,thereby rendering the hapten immunogenic. A representative, non-limitingclass of carriers is proteins, examples of which include albumin,keyhole limpet hemocyanin, hemaglutanin, tetanus, and diptheria toxoid.Other classes and examples of carriers suitable for use in accordancewith the invention are known in the art. These, as well as laterdiscovered or invented naturally occurring or synthetic carriers, can beadapted for application in accordance with the invention.

The term “chemotherapeutic agent” means anti-cancer and otheranti-hyperproliferative agents. Put simply, a “chemotherapeutic agent”refers to a chemical intended to destroy cells and tissues. Such agentsinclude, but are not limited to: DNA damaging agents and agents thatinhibit DNA synthesis: anthracyclines (doxorubicin, donorubicin,epirubicin), alkylating agents (bendamustine, busulfan, carboplatin,carmustine, chlorambucil, cyclophosphamide, dacarbazine,hexamethylmelamine, ifosphamide, lomustine, mechlorethamine, melphalan,mitotane, mytomycin, pipobroman, procarbazine, streptozocin, thiotepa,and triethylenemelamine), platinum derivatives (cisplatin, carboplatin,cis diammine-dichloroplatinum), and topoisomerase inhibitors(Camptosar); anti-metabolites such as capecitabine,chlorodeoxyadenosine, cytarabine (and its activated form, ara-CMP),cytosine arabinoside, dacabazine, floxuridine, fludarabine,5-fluorouracil, 5-DFUR, gemcitabine, hydroxyurea, 6-mercaptopurine,methotrexate, pentostatin, trimetrexate, 6-thioguanine);anti-angiogenics (bevacizumab, thalidomide, sunitinib, lenalidomide,TNP-470, 2-methoxyestradiol, ranibizumab, sorafenib, erlotinib,bortezomib, pegaptanib, endostatin); vascular disrupting agents(flavonoids/flavones, DMXAA, combretastatin derivatives such as CA4DP,ZD6126, AVE8062A, etc.); biologics such as antibodies (Herceptin,Avastin, Panorex, Rituxin, Zevalin, Mylotarg, Campath, Bexxar, Erbitux);endocrine therapy: aromatase inhibitors (4-hydroandrostendione,exemestane, aminoglutehimide, anastrazole, letozole), anti-estrogens(Tamoxifen, Toremifine, Raoxifene, Faslodex), steroids such asdexamethasone; immuno-modulators: cytokines such as IFN-beta and IL2),inhibitors to integrins, other adhesion proteins and matrixmetalloproteinases); histone deacetylase inhibitors like suberoylanilidehydroxamic acid; inhibitors of signal transduction such as inhibitors oftyrosine kinases like imatinib (Gleevec); inhibitors of heat shockproteins like 17-N-allylamino-17-demethoxygeldanamycin; retinoids suchas all trans retinoic acid; inhibitors of growth factor receptors or thegrowth factors themselves; anti-mitotic compounds and/ortubulin-depolymerizing agents such as the taxoids (paclitaxel,docetaxel, taxotere, BAY 59-8862), navelbine, vinblastine, vincristine,vindesine and vinorelbine; anti-inflammatories such as COX inhibitorsand cell cycle regulators, e.g., check point regulators and telomeraseinhibitors.

The term “combination therapy” refers to a therapeutic regimen thatinvolves the provision of at least two distinct therapies to achieve anindicated therapeutic effect. For example, a combination therapy mayinvolve the administration of two or more chemically distinct activeingredients, for example, a fast-acting chemotherapeutic agent and ananti-lipid antibody. Alternatively, a combination therapy may involvethe administration of an anti-lipid antibody and/or one or morechemotherapeutic agents, alone or together with the delivery of anothertreatment, such as radiation therapy and/or surgery. In the context ofthe administration of two or more chemically distinct activeingredients, it is understood that the active ingredients may beadministered as part of the same composition or as differentcompositions. When administered as separate compositions, thecompositions comprising the different active ingredients may beadministered at the same or different times, by the same or differentroutes, using the same of different dosing regimens, all as theparticular context requires and as determined by the attendingphysician. Similarly, when one or more anti-lipid antibody species, forexample, an anti-LPA antibody, alone or in conjunction with one or morechemotherapeutic agents are combined with, for example, radiation and/orsurgery, the drug(s) may be delivered before or after surgery orradiation treatment.

A “derivatized bioactive lipid conjugate” refers to a derivatizedbioactive lipid covalently conjugated to a carrier. The carrier may be aprotein molecule or may be a moiety such as polyethylene glycol,colloidal gold, adjuvants or silicone beads. A derivatized bioactivelipid conjugate may be used as an immunogen for generating an antibodyresponse according to the instant invention, and the same or a differentbioactive lipid conjugate may be used as a detection reagent fordetecting the antibody thus produced. In some embodiments thederivatized bioactive lipid conjugate is attached to a solid supportwhen used for detection.

An “epitope” or “antigenic determinant” refers to that portion of anantigen that reacts with an antibody antigen-binding portion derivedfrom an antibody.

A “hapten” is a substance that is non-immunogenic but can react with anantibody or antigen-binding portion derived from an antibody. In otherwords, haptens have the property of antigenicity but not immunogenicity.

The term “hyperproliferative disorder” refers to diseases and disordersassociated with, the uncontrolled proliferation cells, including but notlimited to uncontrolled growth of organ and tissue cells resulting incancers and benign tumors. Hyperproliferative disorders associated withendothelial cells can result in diseases of angiogenesis such asangiomas, endometriosis, obesity, age-related macular degeneration andvarious retinopathies, as well as the proliferation of endothelial cellsand smooth muscle cells that cause restenosis as a consequence ofstenting in the treatment of atherosclerosis. Hyperproliferativedisorders involving fibroblasts (i.e., fibrogenesis) include but are notlimited to disorders of excessive scarring (i.e., fibrosis) such asage-related macular degeneration, cardiac remodeling and failureassociated with myocardial infarction, excessive wound healing such ascommonly occurs as a consequence of surgery or injury, keloids, andfibroid tumors and stenting.

An “immunogen” is a molecule capable of inducing a specific immuneresponse, particularly an antibody response in an animal to whom theimmunogen has been administered. In the instant invention, the immunogenis a derivatized bioactive lipid conjugated to a carrier, i.e., a“derivatized bioactive lipid conjugate”. The derivatized bioactive lipidconjugate used as the immunogen may be used as capture material fordetection of the antibody generated in response to the immunogen. Thusthe immunogen may also be used as a detection reagent. Alternatively,the derivatized bioactive lipid conjugate used as capture material mayhave a different linker and/or carrier moiety from that in theimmunogen.

To “inhibit,” particularly in the context of a biological phenomenon,means to decrease, suppress or delay. For example, a treatment yielding“inhibition of tumorigenesis” may mean that tumors do not form at all,or that they form more slowly, or are fewer in number than in theuntreated control.

In the context of this invention, a “liquid composition” refers to onethat, in its filled and finished form as provided from a manufacturer toan end user (e.g., a doctor or nurse), is a liquid or solution, asopposed to a solid. Here, “solid” refers to compositions that are notliquids or solutions. For example, solids include dried compositionsprepared by lyophilization, freeze-drying, precipitation, and similarprocedures.

“Monotherapy” refers to a treatment regimen based on the delivery of onetherapeutically effective compound, whether administered as a singledose or several doses over time.

“Neoplasia” refers to abnormal and uncontrolled cell growth. A“neoplasm”, or tumor, is an abnormal, unregulated, and disorganizedproliferation of cell growth, and is generally referred to as cancer. Aneoplasm may be benign or malignant. A neoplasm is malignant, orcancerous, if it has properties of destructive growth, invasiveness, andmetastasis. Invasiveness refers to the local spread of a neoplasm byinfiltration or destruction of surrounding tissue, typically breakingthrough the basal laminas that define the boundaries of the tissues,thereby often entering the body's circulatory system. Metastasistypically refers to the dissemination of tumor cells by lymphatic orblood circulating systems. Metastasis also refers to the migration oftumor cells by direct extension through serous cavities, or subarachnoidor other spaces. Through the process of metastasis, tumor cell migrationto other areas of the body establishes neoplasms in areas away from thesite of initial appearance.

A “patentable” composition, process, machine, or article of manufactureaccording to the invention means that the subject matter satisfies allstatutory requirements for patentability at the time the analysis isperformed. For example, with regard to novelty, non-obviousness, or thelike, if later investigation reveals that one or more claims encompassone or more embodiments that would negate novelty, non-obviousness,etc., the claim(s), being limited by definition to “patentable”embodiments, specifically exclude the non-patentable embodiment(s).Also, the claims appended hereto are to be interpreted both to providethe broadest reasonable scope, as well as to preserve their validity.Furthermore, the claims are to be interpreted in a way that (1)preserves their validity and (2) provides the broadest reasonableinterpretation under the circumstances, if one or more of the statutoryrequirements for patentability are amended or if the standards changefor assessing whether a particular statutory requirement forpatentability is satisfied from the time this application is filed orissues as a patent to a time the validity of one or more of the appendedclaims is questioned.

The term “pharmaceutically acceptable salt” refers to salts which retainthe biological effectiveness and properties of the agents and compoundsof this invention and which are not biologically or otherwiseundesirable. In many cases, the agents and compounds of this inventionare capable of forming acid and/or base salts by virtue of the presenceof charged groups, for example, charged amino and/or carboxyl groups orgroups similar thereto. Pharmaceutically acceptable acid addition saltsmay be prepared from inorganic and organic acids, while pharmaceuticallyacceptable base addition salts can be prepared from inorganic andorganic bases. For a review of pharmaceutically acceptable salts (seeBerge, et al. (1977) J. Pharm. Sci., vol. 66, 1-19).

A “plurality” means more than one.

The terms “separated”, “purified”, “isolated”, and the like mean thatone or more components of a sample contained in a sample-holding vesselare or have been physically removed from, or diluted in the presence of,one or more other sample components present in the vessel. Samplecomponents that may be removed or diluted during a separating orpurifying step include, chemical reaction products, non-reactedchemicals, proteins, carbohydrates, lipids, and unbound molecules.

The term “species” is used herein in various contexts, e.g., aparticular species of chemotherapeutic agent. In each context, the termrefers to a population of chemically indistinct molecules of the sortreferred in the particular context.

“Specifically associate,” “specifically bind” and the like refer to aspecific, non-random interaction between two molecules, whichinteraction depends on the presence of structural,hydrophobic/hydrophilic, and/or electrostatic features that allowappropriate chemical or molecular interactions between the molecules. Anantibody may be said to “bind” or be “reactive with” (or, equivalently,“reactive against”) the epitope of its target antigen. Antibodies arecommonly described in the art as being “against” or “to” their antigensas shorthand for antibody binding to the antigen.

Herein, “stable” refers to an interaction between two molecules (e.g., apeptide and a TLR molecule) that is sufficiently stable such that themolecules can be maintained for the desired purpose or manipulation. Forexample, a “stable” interaction between a peptide and a TLR moleculerefers to one wherein the peptide becomes and remains associated with aTLR molecule for a period sufficient to achieve the desired effect.

A “subject” or “patient” refers to an animal in need of treatment thatcan be effected by molecules of the invention. Animals that can betreated in accordance with the invention include vertebrates, withmammals such as bovine, canine, equine, feline, ovine, porcine, andprimate (including humans and non-humans primates) animals beingparticularly preferred examples.

A “surrogate marker” refers to laboratory measurement of biologicalactivity within the body that indirectly indicates the effect oftreatment on disease state. Examples of surrogate markers forhyperproliferative and/or cardiovascular conditions include SPHK and/orS1PRs.

A “therapeutically effective amount” (or “effective amount”) refers toan amount of an active ingredient, e.g., an agent according to theinvention, sufficient to effect treatment when administered to a subjectin need of such treatment. Accordingly, what constitutes atherapeutically effective amount of a composition according to theinvention may be readily determined by one of ordinary skill in the art.In the context of cancer therapy, a “therapeutically effective amount”is one that produces an objectively measured change in one or moreparameters associated with cancer cell survival or metabolism, includingan increase or decrease in the expression of one or more genescorrelated with the particular cancer, reduction in tumor burden, cancercell lysis, the detection of one or more cancer cell death markers in abiological sample (e.g., a biopsy and an aliquot of a bodily fluid suchas whole blood, plasma, serum, urine, etc.), induction of inductionapoptosis or other cell death pathways, etc. Of course, thetherapeutically effective amount will vary depending upon the particularsubject and condition being treated, the weight and age of the subject,the severity of the disease condition, the particular compound chosen,the dosing regimen to be followed, timing of administration, the mannerof administration and the like, all of which can readily be determinedby one of ordinary skill in the art. It will be appreciated that in thecontext of combination therapy, what constitutes a therapeuticallyeffective amount of a particular active ingredient may differ from whatconstitutes a therapeutically effective amount of the active ingredientwhen administered as a monotherapy (i.e., a therapeutic regimen thatemploys only one chemical entity as the active ingredient).

The term “treatment” or “treating” means any treatment of a disease ordisorder, including preventing or protecting against the disease ordisorder (that is, causing the clinical symptoms not to develop);inhibiting the disease or disorder (i.e., arresting, delaying orsuppressing the development of clinical symptoms; and/or relieving thedisease or disorder (i.e., causing the regression of clinical symptoms).As will be appreciated, it is not always possible to distinguish between“preventing” and “suppressing” a disease or disorder because theultimate inductive event or events may be unknown or latent.Accordingly, the term “prophylaxis” will be understood to constitute atype of “treatment” that encompasses both “preventing” and“suppressing”. The term “protection” thus includes “prophylaxis”.

The term “therapeutic regimen” means any treatment of a disease ordisorder using chemotherapeutic and cytotoxic agents, radiation therapy,surgery, gene therapy, DNA vaccines and therapy, siRNA therapy,anti-angiogenic therapy, immunotherapy, bone marrow transplants,aptamers and other biologics such as antibodies and antibody variants,receptor decoys and other protein-based therapeutics.

SUMMARY OF THE INVENTION

The object of this invention is to provide patentable compositions andmethods for generating antibodies, particularly monoclonal antibodiesand derivatives thereof, reactive with bioactive lipids correlated,involved, or otherwise implicated in disease processes in animals,particularly in mammals, especially humans.

Thus, one aspect of the invention concerns patentable intermediates usedto produce patentable immunogens that can be used to raise patentablebioactive lipid-reactive antibodies. This patentable class of compoundscomprises derivatized bioactive lipids, each of which comprises abioactive lipid having a polar head group and at least one hydrocarbonchain, wherein a carbon atom within the hydrocarbon chain is derivatizedwith a pendant reactive group [e.g., a sulfhydryl (thiol) group, acarboxylic acid group, a cyano group, an ester, a hydroxy group, analkene, an alkyne, an acid chloride group or a halogen atom] that may ormay not be protected. Representative bioactive lipids includelysolipids, for example, sphingolipids and sphingolipid metabolites suchas ceramide, ceramide-1-phosphate, N-acetyl-ceramide-1-phosphate,sphingosine-1-phosphate (S1P), sphingosine, sphingosylphosphorylcholine(SPC), dihydrosphingosine and dihydrosphingosine-1-phosphate. Otherbioactive lipids include lysolipids such as lysophosphatidic acids(LPAs), as well as lysophosphatidic acid metabolites or precursors suchas lysophosphatidylinositol (LPI) or lysophosphatidylcholine (LPC). Inthe context of an LPA, exemplary reactive group positioning includesappending the reactive group to a carbon atom within the hydrocarbonchain or at the sn-1 position of the glycerol backbone of thelysophosphatidic acid moiety. Particularly preferred derivatizedbioactive lipids include sulfhydryl derivatives of LPA and S1P.

A related aspect of the invention relates to immunogens produced from aderivatized bioactive lipid according to the invention. In general, suchimmunogens comprise a derivatized bioactive lipid covalently linked to acarrier. Examples of suitable carrier moieties include carrier proteinssuch as keyhole limpet hemocyanin (KLH) and albumin, polyethyleneglycol, colloidal gold, adjuvants or silicone beads. Preferredembodiments of an immunogen according to the invention include asulfhydryl derivative of LPA covalently linked to KLH or albumin. In thecontext of sphingolipid-based immunogen, preferred immunogen embodimentsinclude sulfhydryl derivatives of S1P covalently linked to KLH oralbumin.

Immunogens of the invention are prepared by reaction of a derivatizedbioactive bioactive lipid with a carrier moiety under conditions thatallow covalent linkage between the carrier and the bioactive lipid tooccur through the pendant reactive group to yield the particular speciesof bioactive lipid-carrier immunogen. Such immunogens are thenpreferably isolated or purified prior to administration to a host animalas part of an immunization procedure, which may involve one or severaladministrations (typically by injection) of the desired immunogen. Inpreferred embodiments of this aspect, the pendant reactive group of thederivatized bioactive lipid is protected with a suitable protectinggroup, which is removed and the derivatized bioactive lipid is“deprotected” prior to or as part of the chemistry employed tocovalently link the carrier and the bioactive lipid.

As discussed above, another aspect of the invention concerns methods ofmaking monoclonal antibody reactive against a bioactive lipid. In suchmethods, an immune competent host animal (e.g., a rodent such as amouse, a rat, a guinea pig, or rabbit) is immunized with a bioactivelipid immunogen as described herein. Following immunization, the hostmounts an antibody response against the bioactive lipid, resulting inthe production of antibodies reactive to the particular bioactive lipidspecies present in the immunogen. The resultant antibodies may bepolyclonal or, preferably, monoclonal. With regard to monoclonalantibodies, cell lines that produce a desired antibody are preferablycloned and immortalized to facilitate production of the desiredlipid-specific antibody in desired quantities. In preferred embodiments,a desired monoclonal antibody, e.g., a monoclonal antibody reactiveagainst LPA is used to produce antibody derivatives, such as chimeric orhumanized antibodies or antibody fragments. In some embodiments, fullyhumanized antibodies may be produced by immunizing an animal, e.g., amouse or rat, engineered to contain some or all of a competent humansystem.

It is known that lipids are in general a particularly intractable classof molecules for antibody production. One facet of the invention restson the appreciation that this problem, at least in part, resides in thedifficulty in detecting antibodies reactive against a particular targetlipid species. However, this problem can be elegantly overcome throughthe use of the derivatized form of the particular target bioactivelipid, such as a lysolipid or a sphingolipid or sphingolipidmetabolite).

In certain preferred embodiments, such a derivatized bioactive lipid maybe used to identify an antibody reactive against an epitope of theparticular bioactive lipid present in the immunogen used to generate theantibodies being detected. To perform this role a particular derivatizedbioactive lipid or derivatized bioactive lipid conjugate may be attachedto a solid support, preferably the solid phase of an assay device, suchas an ELISA plate, a Biacore chip, etc. Attachment to a solid supportminimizes the likelihood that the bioactive lipid will be washed awayduring antibody binding and detection.

Another aspect of the invention concerns pharmaceutical or veterinarycompositions that comprise a carrier and an isolated immune-derivedmoiety according to the invention, for example, a monoclonal antibody orantibody fragment, variant, or derivative. Preferred carriers includethose that are pharmaceutically acceptable, particularly when thecomposition is intended for therapeutic use in humans. For non-humantherapeutic applications (e.g., in the treatment of companion animals,livestock, fish, or poultry), acceptable carriers for veterinary use maybe employed.

Related aspects of the invention relate to methods of use or treatment,including preventative or prophylactic treatment, and administration.Such methods typically involve administering to a subject (for example,mammal, particularly a human patient) in need of therapeutic orprophylactic treatment an amount of an immune-derived moiety reactiveagainst a bioactive lipid target, effective to accomplish the desiredtreatment. In some embodiments the bioactive lipid target is anon-sphingoid bioactive lipid. One preferred example of atherapeutically useful immune-derived moiety is a humanized monoclonalantibody reactive against a lysolipid such as LPA. Routes ofadministration of an immune-derived moiety according to the invention,preferably as part of a therapeutic composition, may vary depending uponwhether local or systemic treatment is desired and upon the area to betreated. Administration may be topical (including transdermal,ophthalmic and to mucous membranes including vaginal, intrauterine andrectal delivery, pulmonary delivery, intratracheal, intranasal, andepidermal delivery), oral or parenteral. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; or intracranial, e.g., intrathecalor intraventricular, administration.

Other aspects of the invention concern various diagnostic, prognostic,and/or research-enabling methods. One such aspect involves use of thederivatized lipid analog to detect the presence of autoantibodiesagainst the natural bioactive lipid in a sample of fluid or tissue froman animal or from an antibody library. Another such aspect concernsmethods of detecting target bioactive lipids, other than sphingolipidsor metabolites thereof. In general, such methods involve binding of animmune-derived moiety with the target bioactive lipid against which itis reactive. Detection of binding may result, for example, by exposing asample (e.g., a biopsy or fluid or liquid sample, for instance, blood,serum, plasma, urine, saliva, tears, cerebrospinal fluid, cell culture,etc.) known or suspected to contain the target bioactive lipid with animmune-derived moiety under conditions that allow the immune-derivedmoiety to bind to the target bioactive lipid, if present in the sample.

To perform such diagnostic methods, reagents are required, anddiagnostic reagents that employ a derivatized lipid according to theinvention represent another aspect of the invention. With such reagentsin hand, diagnostic assays that utilize such reagents may be prepared.

These and other aspects and embodiments of the invention are discussedin greater detail in the sections that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Organic synthesis scheme for making of a typical thiolated-S1Panalog that was used as a key component of an immunogen according to theinvention, as well as a key component of the laydown material for theELISA and BiaCore assays. FIG. 1 a shows the initial portion of asynthesis scheme for a typical thiolated S1P-related antigen. FIGS. 1 band 1 c are continuations of FIG. 1 a showing the remainder of the samesynthetic scheme.

FIG. 2. Organic synthesis scheme for making the thiolated-related fattyacid used in the synthesis of the thiolated-LPA analog of FIG. 3. FIG. 2a is the initial portion of a synthesis scheme for a typical protectedthiolated fatty acid and FIG. 2 b is a continuation of the same showingthe remainder of the scheme.

FIG. 3. Organic synthesis scheme for making the thiolated-LPA analogthat is a key component of an immunogen according to the invention, aswell as a key component of the laydown material for the ELISA and otherassays. FIG. 3 a is the initial portion of a synthesis scheme for atypical thiolated LPA hapten and FIG. 3 b is a continuation of the sameshowing the remainder of the scheme.

FIG. 4. The anti-S1P mAb is specific and sensitive for S1P and does notrecognize structurally similar bioactive lipids. Panel A. CompetitiveELISA with S1P, SPH, LPA, SPC and other structurally similar biolipidscompeting for the mAb binding to S1P on the plate. Only free S1P orDH-S1P can compete for binding, demonstrating the specificity of theanti-S1P mAb. SPC only slightly competes for binding. Panel B.Structures of bioactive lipids used in the evaluation of specificity.

FIG. 5. BiaCore analysis of binding kinetics of anti-S1P mAb to thio-S1Ptethered to a Biacore maleimide surface CM5 sensor chip. Variousdilutions of anti-S1P mAb were applied to the flow cell for generatingsensograms.

FIG. 6. Amino acid sequences of the mouse V_(H) (SEQ ID NO: 6) and V_(L)(SEQ ID NO: 9) domains of murine Sphingomab™. CDR residues are boxed.

FIG. 7. Nucleotide and amino acid sequences of the V_(H) and V_(L)domains of murine Sphingomab™ (SEQ ID NOs: 16-46 as indicated).

FIG. 8. Graph showing ELISA results for binding studies of murineSphingomab™ and chimeric, SIP-binding antibodies derived from murineSphingomab™.

FIG. 9. Direct ELISA showing binding of murine and chimeric mAbs toELISA plates coated with thiolated S1P analog as described in EXAMPLE 6.Data show that the chimeric mAb (cα-S1P IgG) has similar, if not greaterbinding performance compared to the fully murine mAb (mα-S1P IgG).

As those in the art will appreciate, the following description describescertain preferred embodiments of the invention in detail, and is thusonly representative and does not depict the actual scope of theinvention. Before describing the present invention in detail, it isunderstood that the invention is not limited to the particularmolecules, systems, and methodologies described, as these may vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the invention defined by the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for generatingand identifying antibodies against bioactive lipid molecules that play arole in human and/or animal disease as a signaling molecule. Theinvention also relates to these antibodies themselves, and methods ofusing them therapeutically, diagnostically and as research reagents.

1. Methods for Antibody Production and Identification

It is known that lipids are in general a particularly intractable classof molecules for antibody production. Antibody production can typicallybe described as a two-part process: a suitable immunogen must beprovided which will generate the desired antibody response in an animal,and the resulting antibody, if present, must be detectable.

As discussed above, effective antibody production requires both antibodygeneration and antibody detection. As disclosed in the Exampleshereinbelow, generation of antibodies targeted to certain bioactivelipids has been achieved using derivatized bioactive lipid as immunogen.In the examples, the thiolated bioactive lipid (e.g., S1P) analog wasconjugated to Keyhole Limpet Hemocyanin (KLH) or to fatty-acid freeBovine Serum Albumin (BSA) via SMCC (Pierce, Rockford Ill.) usingprotocols recommended by the manufacturer. SMCC is a heterobifunctionalcrosslinker that reacts with primary amines and sulfhydryl groups, andrepresents a preferred crosslinker. Iodoacetamide (IOA) can also be usedfor maleimide-activated proteins.

However, other immunogens and methods of generating antibodies known inthe art may also be used. For example, antibodies against phospholipidspecies have been generated by immunization with liposomes(Maneta-Peyret et al., 1988, 1989; Benerji and Alving, 1990) or byadsorption of monomeric phospholipids to proteins (Tamamura et al.,1971; Maneta-Peyret et al., 1989), to bacteria (Umeda et al., 1989), toacrylamide (Maneta-Peyret et al., 1988, 1989) and to gold [Tomii et al.,(1991) Jpn J. Med. Sci. Biol. 44:75-80]. In many cases, presentation ofthe bioactive lipid as emulsions or liposomal complexes has resulted inIgMs with limited specificity, sensitivity and/or biological activity incomparison to IgG. For example, two commercially available reagentssupposedly specific for ceramide, one an IgM-enriched polyclonal mouseserum and the other an IgM monoclonal antibody, were characterized. Themonoclonal was found to be specific for sphingomyelin and the antiserumwas found to react with various ceramide species in the nanomolar range.Vielhaber, G. et al., (2001) Glycobiology 11:451-457. In a differentapproach, Ran et al. [(2005) Clin. Cancer Res. 11: 1551-1562] usedb.End3 endothelial cells that had been treated with peroxide (intendedto cause translocation of anionic phospholipids to the external surfaceof the cells) as an immunogen to elicit generation of antibodiesspecific for anionic phospholipids. Thus numerous methods are known bywhich an antibody response to a desired antigenic target may beelicited; any of these may be used in the instant invention as long asthe resulting antibodies can be detected and shown to be reactive withthe desired bioactive lipid.

Antibody generation, while of course necessary, is not sufficient if theantibody cannot be detected. Thus one facet of the invention rests onthe appreciation that previous failures of others to produce antibodiesto bioactive lipids may be attributable at least to shortcomings in thedetection step. This problem of detection has been elegantly overcome inthe following examples through the use of a derivatized bioactive lipid.The derivatized bioactive lipid is used to detect and identify anantibody reactive against an epitope of the particular bioactive lipidpresent in the immunogen used to generate the antibodies being detected;the bioactive lipid used for detection in derivatized form contains thesame epitope to which antibodies were generated. To perform this rolethe derivatized lipid may be associated with the solid phase of an assaydevice, such as an ELISA plate, a BiaCore sensor chip, etc. In someembodiments the derivatized bioactive lipid is covalently conjugateddirectly to the solid support. By way of example, the derivatized lipidmay be covalently conjugated to an activated BiaCore chip as describedin Examples hereinbelow. In other embodiments, the derivatized bioactivelipid is covalently conjugated to a carrier moiety, yielding a“derivatized bioactive lipid conjugate” which is then bound to a solidsupport. As an example, derivatized lipid covalently conjugated to BSAis used as the laydown material (capture material) for ELISA asdescribed in Examples hereinbelow. In either embodiment, attachment ofthe derivatized bioactive lipid to the solid support provides a stabledetection means which is unlikely to be washed away, as is a risk ofsome detection methods. Detection of the antibody may be accomplished ina variety of ways. In a preferred embodiment of the invention, thedetection is via ELISA, Biacore™ label-free interaction analysissystems, or other solid-support-based routine detection means in whichthe derivatized bioactive lipid is attached to said solid support.Examples of other solid supports include but are not limited to affinitycolumns, glass or synthetic beads, multiwell plates and the like.

The derivatized bioactive lipid conjugate used in the detection step maybe the same derivatized bioactive lipid conjugate used as the immunogen,or the derivatized bioactive lipid may be conjugated to a differentcarrier than in the conjugate used as the immunogen. In someembodiments, e.g. as the laydown for ELISA, it is preferred to use adifferent derivatized bioactive lipid conjugate in the detection step,than was used as the immunogen, to minimize crossreactivity. By way ofexamples, the carrier may be BSA (preferably fatty-acid free,particularly in the detection step), KLH or other carriers known in theart. The crosslinker used to conjugate the derivatized bioactive lipidto the protein carrier may be, for example, SMCC or IOA. In onepreferred embodiment the immunogen is S1P-IOA-KLH and S1P-SMCC-BSA(fatty acid free BSA) is the capture laydown material in the ELISA,wherein S1P refers to the derivatized S1P that reacts with thecrosslinker (IOA or SMCC in this instance) to form a covalent bond withthe protein carrier (KLH or BSA in this instance).

2. Compounds

The term “antibody” (“Ab”) or “immunoglobulin” (Ig) refers to any formof a peptide, polypeptide derived from, modeled after or encoded by, animmunoglobulin gene, or fragment thereof, capable of binding an antigenor epitope. See, e.g., IMMUNOBIOLOGY, Fifth Edition, C. A. Janeway, P.Travers, M., Walport, M. J. Shlomchiked., ed. Garland Publishing (2001).Antibody molecules or immunoglobulins are large glycoprotein moleculeswith a molecular weight of approximately 150 kDa, usually composed oftwo different kinds of polypeptide chain. One polypeptide chain, termedthe “heavy” chain (H) is approximately 50 kDa. The other polypeptide,termed the “light” chain (L), is approximately 25 kDa. Eachimmunoglobulin molecule usually consists of two heavy chains and twolight chains. The two heavy chains are linked to each other by disulfidebonds, the number of which varies between the heavy chains of differentimmunoglobulin isotypes. Each light chain is linked to a heavy chain byone covalent disulfide bond. In any given naturally occurring antibodymolecule, the two heavy chains and the two light chains are identical,harboring two identical antigen-binding sites, and are thus said to bedivalent, i.e., having the capacity to bind simultaneously to twoidentical molecules.

The “light” chains of antibody molecules from any vertebrate species canbe assigned to one of two clearly distinct types, kappa (k) and lambda(λ), based on the amino acid sequences of their constant domains. Theratio of the two types of light chain varies from species to species. Asa way of example, the average k to λ ratio is 20:1 in mice, whereas inhumans it is 2:1 and in cattle it is 1:20.

The “heavy” chains of antibody molecules from any vertebrate species canbe assigned to one of five clearly distinct types, called isotypes,based on the amino acid sequences of their constant domains. Someisotypes have several subtypes. The five major classes of immunoglobulinare immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G(IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is themost abundant isotype and has several subclasses (IgG1, 2, 3, and 4 inhumans). The Fc fragment and hinge regions differ in antibodies ofdifferent isotypes, thus determining their functional properties.However, the overall organization of the domains is similar in allisotypes.

As used herein, “antibody fragment” and grammatical variants thereofrefer to a portion of an intact antibody that includes the antigenbinding site or variable regions of an intact antibody, wherein theportion can be free of the constant heavy chain domains (e.g., CH2, CH3,and CH4) of the Fc region of the intact antibody. Alternatively,portions of the constant heavy chain domains (e.g., CH2, CH3, and CH4)can be included in the “antibody fragment”. Examples of antibodyfragments are those that retain antigen-binding and include Fab, Fab′,F(ab′)₂, Fd, and Fv fragments; diabodies; triabodies; single-chainantibody molecules (sc-Fv); minibodies, nanobodies, and multispecificantibodies formed from antibody fragments. By way of example, a Fabfragment also contains the constant domain of a light chain and thefirst constant domain (CH1) of a heavy chain.

The term “variable region” refers to N-terminal sequence of the antibodymolecule or a fragment thereof. In general, each of the four chains hasa variable (V) region in its amino terminal portion, which contributesto the antigen-binding site, and a constant (C) region, which determinesthe isotype. The light chains are bound to the heavy chains by manynoncovalent interactions and by disulfide bonds, and the V regions ofthe heavy and light chains pair in each arm of antibody molecule togenerate two identical antigen-binding sites. Some amino acid residuesare believed to form an interface between the light- and heavy-chainvariable domains (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991); Clothia et al., J. Mol. Biol., vol. 186:651(1985)).

Of note, variability is not uniformly distributed throughout thevariable domains of antibodies, but is concentrated in three segmentscalled “complementarity-determining regions” (CDRs) or “hypervariableregions” both in the light-chain and the heavy-chain variable domains.The more highly conserved portions of variable domains are called the“framework region” (FR). The variable domains of native heavy and lightchains each comprise four FR regions connected by three CDRs. The CDRsin each chain are held together in close proximity by the FR regionsand, with the CDRs from the other chain, contribute to the formation ofthe antigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). Collectively, the 6 CDRs contribute tothe binding properties of the antibody molecule. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen (seePluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994)).

The terms “constant domain” refers to the C-terminal region of anantibody heavy or light chain. Generally, the constant domains are notdirectly involved in the binding properties of an antibody molecule toan antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.Here, “effector functions” refer to the different physiological effectsof antibodies (e.g., opsonization, cell lysis, mast cell, basophil andeosinophil degranulation, and other processes) mediated by therecruitment of immune cells by the molecular interaction between the Fcdomain and proteins of the immune system. The isotype of the heavy chaindetermines the functional properties of the antibody. Their distinctivefunctional properties are conferred by the carboxy-terminal portions ofthe heavy chains, where they are not associated with light chains.

The term “variant” refers to an amino acid sequence which differs fromthe native amino acid sequence of an antibody by at least one amino acidresidue modification. A native or parent or wild-type amino acidsequence refers to the amino acid sequence of an antibody found innature. “Variant” of the antibody molecule includes, but is not limitedto, changes within a variable region or a constant region of a lightchain and/or a heavy chain, including in the Fc region, the Fab region,the CH₁ domain, the CH₂ domain, the CH₃ domain, and the hinge region.

The term “specific” refers to the selective binding of an antibody toits target epitope. Antibody molecules can be tested for specificity ofbinding by comparing binding to the desired antigen to binding tounrelated antigen or analogue antigen or antigen mixture under a givenset of conditions. Preferably, an antibody according to the inventionwill lack significant binding to unrelated antigens, or even analogs ofthe target antigen. Here, the term “antigen” refers to a molecule thatis recognized and bound by an antibody molecule or immune-derived moietythat binds to the antigen. The specific portion of an antigen that isbound by an antibody is termed the “epitope”. A “hapten” refers to asmall molecule that can, under most circumstances, elicit an immuneresponse (i.e., act as an antigen) only when attached to a carrier, forexample, a protein, polyethylene glycol (PEG), colloidal gold, siliconebeads, and the like. The carrier may be one that also does not elicit animmune response by itself.

The term “antibody” is used in the broadest sense, and encompassesmonoclonal, polyclonal, multispecific (e.g., bispecific, wherein eacharm of the antibody is reactive with a different epitope of the same ordifferent antigen), minibody, heteroconjugate, diabody, triabody,chimeric, and synthetic antibodies, as well as antibody fragments thatspecifically bind an antigen with a desired binding property and/orbiological activity.

The term “monoclonal antibody” (mAb) refers to an antibody, orpopulation of like antibodies, obtained from a population ofsubstantially homogeneous antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, monoclonal antibodies can be made by the hybridoma method firstdescribed by Kohler G. and Milstein C. (1975), Nature, vol. 256:495-497,or by recombinant DNA methods.

The term “chimeric” antibody (or immunoglobulin) refers to a moleculecomprising a heavy and/or light chain which is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (Cabilly, et al., infra; Morrison et al., Proc.Natl. Acad. Sci. U.S.A., vol. 81:6851 (1984)).

The term “humanized antibody” means human antibodies that also containselected sequences from non-human (e.g., murine) antibodies in place ofthe human sequences. A humanized antibody can include conservative aminoacid substitutions or non-natural residues from the same or differentspecies that do not significantly alter its binding and/or biologicactivity. Such antibodies are chimeric antibodies that contain minimalsequence derived from non-human immunoglobulins. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, camel, bovine, goat, or rabbithaving the desired properties. Furthermore, humanized antibodies cancomprise residues that are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and maximize antibody performance. Thus, in general, ahumanized antibody will comprise all of at least one, and in one aspecttwo, variable domains, in which all or all of the hypervariable loopscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), or that of a humanimmunoglobulin. See, e.g., Cabilly, et al, U.S. Pat. No. 4,816,567;Cabilly, et al., European Patent No. 0,125,023 B1; Boss, et al., U.S.Pat. No. 4,816,397; Boss, et al., European Patent No. 0,120,694 B1;Neuberger, et al., WO 86/01533; Neuberger, et al., European Patent No.0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European PatentNo. 0,239,400 B1; Padlan, et al., European Patent Application No.0,519,596 A1; Queen, et al. (1989), Proc. Nat'l Acad. Sci. USA, vol.86:10029-10033).

The term ‘fully human’ antibody can refer to an antibody produced in agenetically engineered (ie. Transgenic) mouse (e.g. from Medarex) that,when presented with an immunogen, can produce a human antibody that doesnot necessarily require CDR grafting. These antibodies are fully human(100% human protein sequences) from animals such as mice in which thenon-human antibody genes are suppressed and replaced with human antibodygene expression. The applicants believe that antibodies could begenerated against bioactive lipids when presented to these geneticallyengineered mice or other animals who might be able to produce humanframeworks for the relevant CDRs.

The term “bispecific antibody” can refer to an antibody, or a monoclonalantibody, having binding properties for at least two different epitopes.In one embodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs.Alternatively, bispecific antibodies can be prepared using chemicallinkage. One of skill can produce bispecific antibodies using these orother methods as may be known in the art. Bispecific antibodies includebispecific antibody fragments. One example of a bispecific antibodycomprehended by this invention is an antibody having binding propertiesfor an S1P epitope and an LPA epitope, which thus is able to recognizeand bind to both S1P and LP1. Another example of a bispecific antibodycomprehended by this invention is an antibody having binding propertiesfor an epitope from a bioactive lipid and an epitope from a cell surfaceantigen. Thus the antibody is able to recognize and bind the bioactivelipid and is able to recognize and bind to cells, e.g., for targetingpurposes.

The term “heteroconjugate antibody” can refer to two covalently joinedantibodies. Such antibodies can be prepared using known methods insynthetic protein chemistry, including using crosslinking agents. Asused herein, the term “conjugate” refers to molecules formed by thecovalent attachment of one or more antibody fragment(s) or bindingmoieties to one or more polymer molecule(s).

The term “biologically active” refers to an antibody or antibodyfragment that is capable of binding the desired epitope and in some waysexerting a biologic effect. Biological effects include, but are notlimited to, the modulation of a growth signal, the modulation of ananti-apoptotic signal, the modulation of an apoptotic signal, themodulation of the effector function cascade, and modulation of otherligand interactions.

The term “recombinant DNA” refers to nucleic acids and gene productsexpressed therefrom that have been engineered, created, or modified byman. “Recombinant” polypeptides or proteins are polypeptides or proteinsproduced by recombinant DNA techniques, for example, from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or proteins are thoseprepared by chemical synthesis.

The term “expression cassette” refers to a nucleotide molecule capableof affecting expression of a structural gene (i.e., a protein codingsequence, such as an antibody of the invention) in a host compatiblewith such sequences. Expression cassettes include at least a promoteroperably linked with the polypeptide-coding sequence, and, optionally,with other sequences, e.g., transcription termination signals.Additional regulatory elements necessary or helpful in effectingexpression may also be used, e.g., enhancers. Thus, expression cassettesinclude plasmids, expression vectors, recombinant viruses, any form ofrecombinant “naked DNA” vector, and the like.

A “vector” or “plasmid” or “expression vector” refers to a nucleic acidthat can be maintained transiently or stably in a cell to effectexpression of one or more recombinant genes. A vector can comprisenucleic acid, alone or complexed with other compounds. A vectoroptionally comprises viral or bacterial nucleic acids and/or proteins,and/or membranes. Vectors include, but are not limited, to replicons(e.g., RNA replicons, bacteriophages) to which fragments of DNA may beattached and become replicated. Thus, vectors include, but are notlimited to, RNA, autonomous self-replicating circular or linear DNA orRNA and include both the expression and non-expression plasmids.“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids asreported with published protocols. In addition, the expression vectorsmay also contain a gene to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The term “promoter” includes all sequences capable of drivingtranscription of a coding sequence in a cell. Thus, promoters used inthe constructs of the invention include cis-acting transcriptionalcontrol elements and regulatory sequences that are involved inregulating or modulating the timing and/or rate of transcription of agene. For example, a promoter can be a cis-acting transcriptionalcontrol element, including an enhancer, a promoter, a transcriptionterminator, an origin of replication, a chromosomal integrationsequence, 5′ and 3′ untranslated regions, or an intronic sequence, whichare involved in transcriptional regulation. Transcriptional regulatoryregions suitable for use in the present invention include but are notlimited to the human cytomegalovirus (CMV) immediate-earlyenhancer/promoter, the SV40 early enhancer/promoter, the E. coli lac ortrp promoters, and other promoters known to control expression of genesin prokaryotic or eukaryotic cells or their viruses.

A. Antibodies to Sphingolipids

The present invention provides methods for preparing antibodies directedagainst certain bioactive lipids, including sphingolipids. The term“sphingolipid” refers to the sphingolipids as defined byhttp//www.lipidmaps.org, including the following: Sphingoid bases[including sphing-4-enines (sphingosines), sphinganines,4-hydroxysphinganines (phytosphingosines), sphingoid base homologs andvariants, sphingoid base 1-phosphates, lysosphingomyelins andlysoglycosphingolipids; N-methylated sphingoid bases, and sphingoid baseanalogs]; ceramides [including N-acylsphingosines (ceramides),N-acylsphinganines (dihydroceramides), N-acyl-4-hydroxysphinganines(phytoceramides), acylceramides and ceramide 1-phosphates];phosphosphingolipids [including ceramide phosphocholines(sphingomyelins), ceramide phosphoethanolamines and ceramidephosphoinositols; phosphonosphingolipids; neutral glycosphingolipids[including the simple Glc series (GlcCer, LacCer, etc.,GalNAcb1-3Gala1-4Galb1-4Glc- (Globo series), GalNAcb1-4Galb1-4Glc-(Ganglio series), Galb1-3GlcNAcb1-3Galb1-4Glc- (Lacto series),Galb1-4GlcNAcb1-3Galb1-4Glc- (Neolacto series),GalNAcb1-3Gala1-3Galb1-4Glc- (Isoglobo series),GlcNAcb1-2Mana1-3Manb1-4Glc- (Mollu series),GalNAcb1-4GlcNAcb1-3Manb1-4Glc- (Arthro series), Gal- (Gala series) orother neutral glycosphingolipids]; acidic glycosphingolipids [includinggangliosides, sulfoglycosphingolipids (sulfatides),glucuronosphingolipids, phosphoglycosphingolipids and other acidicglycosphingolipids; basic glycosphingolipids; amphotericglycosphingolipids; arsenosphingolipids and other sphingolipids.

Anti-sphingolipid antibodies are useful for treating or preventingdisorders such as hyperproliferative disorders and cardiovascular orcerebrovascular diseases and disorders, as described in greater detailbelow. In particular embodiments the invention is drawn to methods ofpreparing antibodies to S1P and its variants which include S1P itself{defined as sphingosine-1-phosphate [sphingene-1-phosphate;D-erythro-sphingosine-1-phosphate; sphing-4-enine-1-phosphate;(E,2S,3R)-2-amino-3-hydroxy-octadec-4-enoxy]phosphonic acid] (CAS26993-30-6)}, or DHS1P {defined as dihydro sphingosine-1-phosphate[sphinganine-1-phosphate;[(2S,3R)-2-amino-3-hydroxy-octadecoxy]phosphonic acid;D-Erythro-dihydro-D-sphingosine-1-phosphate] (CAS 19794-97-9)}.Antibodies to SPC {defined as sphingosylphosphoryl choline,lysosphingomyelin, sphingosylphosphocholine, sphingosinephosphorylcholine, ethanaminium;2-((((2-amino-3-hydroxy-4-octadecenyl)oxy)hydroxyphosphinyl)oxy)-N,N,N-trimethyl-,chloride, (R—(R*,S*-(E))),2-[[(E,2R,3S)-2-amino-3-hydroxy-octadec-4-enoxy]-hydroxy-phosphoryl]oxyethy1-trimethyl-azanium chloride (CAS 10216-23-6)]} may also be useful.

1. A Preferred Anti-S1P Monoclonal Antibody.

A specific monoclonal anti-S1P antibody (anti-S1P mAb) is described.This antibody can be used as a therapeutic molecular sponge toselectively absorb S1P and thereby thus lower the effective in vivoextracellular concentrations of this pro-angiogenic, pro-fibrotic andtumor-facilitating factor. This can result in the reduction of tumorvolume and metastatic potential, as well as the simultaneous blockage ofnew blood vessel formation that otherwise can feed the growing tumor.This antibody (and molecules having an equivalent activity) can also beused to treat other hyperproliferative disorders impacted by S1P,including unwanted endothelial cell proliferation, as occurs, forexample, in age-related macular degeneration as well as in many cancers.In addition, the ability of S1P to protect cells from apoptosis can bereversed by the agents such as the antibody resulting in an increase inthe efficacy of standard pro-apoptotic chemotherapeutic drugs.

B. Antibodies to Other Bioactive Signaling Lipids

The methods described herein can be used to prepare monoclonalantibodies against many additional extracellular and intracellularbioactive lipids beyond sphingolipids (e.g., SPC, ceramide, sphingosine,sphinganine, S1P and dihydro-S1P). Other bioactive lipid classes includethe leukotrienes, eicosanoids, eicosanoid metabolites such as the HETEs,prostaglandins, lipoxins, epoxyeicosatrienoic acids and isoeicosanoids),non-eicosanoid cannabinoid mediators, phospholipids and theirderivatives such as phosphatidic acid (PA) and phosphatidylglycerol(PG), cardiolipins, and lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA). In short, this inventioncan be adapted for application to any desired extracellular and/orintracellular signaling bioactive lipid with pleiotropic effects onimportant cellular processes. Other examples of bioactive lipids includephosphatidylinositol (PI), phosphatidylethanolamine (PEA),diacylglyceride (DG), sulfatides, gangliosides, globosides andcerebrosides.

C. Conjugates.

A monoclonal antibody, or antigen-binding fragment thereof, describedherein can be used alone in vitro or can be administered to a subject,in non-derivatized or non-conjugated forms. In other embodiments, suchantibodies, derivatives, and variants can be derivatized or linked toone or more molecular entities. Other molecular entities includenaturally occurring, recombinant, or synthetic peptides, polypeptides,and proteins, nonpeptide chemical compounds such as isotopes, smallmolecule therapeutics, etc. Preferred small molecules includeradiolabels, fluorescent agents, and small molecule chemotherapeuticagents. Preferred proteins include growth factors, cytokines, andantibodies (including identical antibodies and derivatives or variantsof such antibodies). The active ingredients can be linked by anysuitable method, taking into account the active ingredients and theintended application, among other factors. For example, a monoclonalantibody of the invention can be functionally linked to another moleculeby chemical coupling, genetic fusion, non-covalent association, oranother suitable approach.

The invention thus envisions conjugates formed between one or moremonoclonal antibodies of the invention, or a variant or derivativethereof, with another active ingredient. Such conjugates may be covalentor non-covalent, and may occur via a linker or directly between theactive ingredients. Examples of such conjugates include one or moremonoclonal antibodies of the invention (or an antigen-binding domainthereof) linked to another therapeutic monoclonal antibody of the sameor different class. Alternatively, the monoclonal antibody or antibodyderivative or variant of the invention may be linked to a differentclass of therapeutic agent, for example, a small moleculechemotherapeutic agent or radioisotope. In some embodiments, one or moreof each of two or more different therapeutic agents (at least one ofwhich is a compound of the invention) can be linked through amultivalent scaffold.

As an alternative to conjugates, a monoclonal antibody or antibodyderivative or variant of the invention may simply be associated with oneor more different therapeutic agents. As an example, a monoclonalantibody of the invention can be combined with one or more other typesof therapeutic agents in a delivery vehicle, e.g., a liposome, micelle,nanoparticle, etc., suitable for administration to a subject.

The invention also envisions conjugating a monoclonal antibody orantibody derivative or variant of the invention, for example, one ormore CDRs reactive against a particular target bioactive lipid, with aprotein or polypeptide. As an example, one or more CDRs from thevariable region of a immunoglobulin heavy or light chain can be graftedinto monoclonal antibody.

3. Applications

The invention is drawn to compositions and methods for treating orpreventing hyperproliferative disorders such as cancer, fibrosis andangiogenesis, and cardiovascular, cardiac, and other diseases, disordersor physical trauma, and/or cerebrovascular diseases and disorders, inwhich therapeutic agents are administered to a patient that alters theactivity or concentration of an undesirable, toxic and/or bioactivelipids, or precursors or metabolites thereof. The therapeutic methodsand compositions of the invention act by changing the absolute, relativeand/or available concentration and/or activities of certain undesirableor toxic lipids. Here, “toxic” refers to a particular lipid'sinvolvement in a disease process, for example, as a signaling molecule.

Without wishing to be bound by any particular theory, it is believedthat inappropriate concentrations of lipids such as LPA and/or theirmetabolites cause or contribute to the development of various diseasesand disorders, including heart disease, neuropathic pain, cancer,angiogenesis, inflammation, and cerebrovascular disease, includingstroke-like inner ear pathologies (see, e.g., Scherer, et al. (2006),Cardiovascular Research, vol. 70; 79-87). As such, the instantcompositions and methods can be used to treat these diseases anddisorders, particularly by decreasing the effective in vivoconcentration of a particular target lipid, for example, LPA. Severalclasses of diseases that may be treated in accordance with the inventionare described below.

A. Hyperproliferative Diseases and Disorders

i. Cancer

One cancer therapy strategy is to reduce the biologically availableextracellular levels of the tumor-promoter, S1P, either alone or incombination with traditional anti-cancer treatments, including theadministration of chemotherapeutic agents, such as an anthracycline. Tothis end, a monoclonal antibody (mAb) has been developed that isspecific for S1P, which can selectively adsorb S1P from the serum,acting as a molecular sponge to neutralize extracellular S1P. Since S1Phas been shown to be pro-angiogenic, an added benefit to the antibody'seffectiveness can be derived from the antibody's ability to starve theblood supply of the growing tumor. Thus, another sphingolipid-basedanti-neoplastic strategy involves combining known activators of CER andSPH production (doxorubicin and related anthracycline glycosides,radiation therapy, etc.) coupled with a strategy to reduce S1P levels.

While sphingolipid-based anti-cancer strategies that target key enzymesof the sphingolipid metabolic pathway, such as SPHK, have been proposed,S1P itself has not been emphasized, largely because of difficulties inattacking this and related targets. As described herein, a highlyspecific monoclonal antibody to S1P has been produced that recognizesS1P in the physiological range and is capable of neutralizing S1P bymolecular combination. Use of this antibody (and its derivatives) willdeprive growing tumor cells of an important growth and survival factor.Moreover, use of such an antibody-based cancer therapy could also beeffective when used in combination with conventional cancer treatments,such as surgery, radiation therapy, and/or the administration ofcytotoxic anti-cancer agents. Examples of cytotoxic agents include, forexample, the anthracycline family of drugs, the vinca alkaloids, themitomycins, the bleomycins, the cytotoxic nucleosides, the taxanes, theepothilones, discodermolide, the pteridine family of drugs, diynenes andthe podophyllotoxins. Members of those classes include, for example,doxorubicin, caminomycin, daunorubicin, aminopterin, methotrexate,methopterin, dichloromethotrexate, mitomycin C, porfiromycin,5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside,podophyllotoxin or podophyllotoxin derivatives, such as etoposide,etoposide phosphate or teniposide, melphalan, vinblastine, vincristine,leurosidine, vindesine, leurosine, paclitaxel and the like. Otherantineoplastic agents include estramustine, cisplatin, carboplatin,cyclophosphamide, bleomycin, gemcitibine, ifosamide, melphalan,hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate,dacarbazine, L-asparaginase, camptothecin, CPT-11, topotecan, ara-C,bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives,interferons and interleukins. Other cytotoxic drugs are well known inthe art. An antibody-based combination therapy may improve the efficacyof chemotherapeutic agents by sensitizing cells to apoptosis whileminimizing their toxic side effects, although administration of theantibody alone may also have efficacy in delaying the progression ofdisease. Indeed, the ability of the anti-S1P mAb to retard tumorprogression in mouse models of human cancer and in allograft mousemodels demonstrates the utility of anti-S1P antibody approaches intreating both human and animal tumors. Moreover, the discovery thatseveral human cancers types (e.g., ovarian, breast, lung, and melanoma)can be treated in xenograft models demonstrates that the anti-S1Pantibody approaches are not limited to one cancer cell or tissue type.

LPA mediates multiple cellular responses including cell proliferation,differentiation, angiogenesis and motility. A large body of experimentalfindings suggests that extracellular LPA plays a key role in theprogression of several types of human cancer by stimulating tumor cellproliferation, survival, invasion and by inducing angiogenesis andmetastasis. In addition, LPA protects a variety of tumor cell types fromapoptosis. LPA has long been associated with ovarian and breast cancer[Fang, X., et al., (2002) Biochim Biophys Acta, 1582: 257-64]; elevatedlevels of LPA have been found in both blood and ascites of patients andhave been correlated with tumor progression, angiogenesis and metastaticpotential. Furthermore, autotoxin (ATX), the enzyme primarilyresponsible for LPA production, has been correlated with the metastaticand invasive properties of human tumors including melanoma, lung cancer,neuroblastoma, hepatocellular carcinoma, and glioblastoma multiforme.Thus LPA is recognized to be an innovative and promising target forcancer therapy [Mills, G. B. and W. H. Moolenaar (2003) Nat Rev Cancer,3: 582-91].

It is believed that neutralizing LPA with anti-LPA antibody (such asthat disclosed herein) will be a novel anti-angiogenic andanti-metastatic therapeutic approach in the treatment of cancer.Monoclonal antibodies against LPA are believed to act as a “sponge” toselectively bind LPA and thereby lower the effective in vivoextracellular levels of LPA. This is believed to result in the reductionof tumorigenesis and tumor growth as well as the simultaneous blockageof blood vessel formation and the metastatic potential. In addition, theability of LPA to protect cells from apoptosis is likely to be lost as aresult of antibody neutralization, thus increasing the efficacy ofstandard pro-apoptotic chemotherapeutic drugs.

ii. Angiogenesis

Angiogenesis is the process by which new blood vessels are formed fromexisting blood vessels. The angiogenesis associated with solid andcirculating tumors is now considered to be a crucial component oftumorigenesis, as today the view that tumor growth is dependent uponneovascularization is scientifically well accepted. Both S1P and LPAappear important to the angiogenic process.

LPA is the primary regulator of GROα, an oncogene believed to contributeto tumorigenesis through its pro-angiogenic effect (Lee, et al (2006),Cancer Res, vol. 66: 2740-8). LPA also enhances expression of matrixmetalloproteinase-2, a recognized player in the cell migrationunderlying the angiogenic process (Wu, et al. (2005), Endocrinology,vol. 146: 3387-3400).

S1P stimulates DNA synthesis and chemotactic motility of human venousendothelial cells (HUVECs), while inducing differentiation ofmulticellular structures essential early blood vessel formation. S1Palso promotes the migration of bone marrow-derived endothelial cellprecursors to neovascularization sites, and cells that over-express S1Preceptors are resistant the anti-angiogenic agents, thalidomide andNeovastat. Thus, S1P, and particularly S1 receptors, are required forangiogenesis and neovascularization. Finally, cross-talk occurs betweenS1P and other pro-angiogenic growth factors such as VEGF, EGF, PDGF,bFGF, and IL-8. For example, S1P transactivates EGF and VEGF2 receptors,and VEGF up-regulates S1P receptor expression (Igarashi, et al. (2003),PNAS (USA), vol. 100: 10664-10669).

As will be appreciated, clinical control of angiogenesis is a criticalcomponent for the treatment of cancer and other angiogenesis-dependentdiseases such as age-related macular degeneration (AMD) andendometriosis. Anti-angiogenic therapeutics are also particularlyattractive because the vascular endothelial cells that are involved intumor angiogenesis do not mutate as easily as do cancer cells;consequently, vascular endothelial cells are less likely than cancercells to gain resistance to prolonged therapy, making them usefultherapeutic targets.

There are several lines of evidence suggesting that S1P is a potentiallysignificant pro-angiogenic growth factor that may be important in tumorangiogenesis, including that: anti-S1P antibodies can neutralizeS1P-induced tube formation, migration of vascular endothelial cells, andprotection from cell death in various in vitro assays using HUVECs;injection of breast adenocarcinoma MCF-7 cells expressing elevated S1Plevels into mammary fat pads of nude mice results in an increase ofangiogenesis-dependent tumors that are both larger and more numerousthan when control cells are used; anti-S1P antibodies can dramaticallyreduce tumor-associated angiogenesis in an orthotopic murine melanomaallograft model; S1P increases new capillary growth into Matrigel plugsimplanted in mice, an effect that can be neutralized by the systemicadministration of anti-S1P antibodies; in vivo administration ofanti-S1P antibodies can completely neutralize pro-angiogenic growthfactor-induced angiogenesis (e.g., by bFGF and VEGF) in murine Matrigelplug assays; S1P stimulates the release of bFGF and VEGF from tumorcells in vitro and in vivo, an effect that can be reversed by anti-S1Pantibodies; S1P enhances in vitro motility and invasion of a largenumber of different types of cancer cells, including glioblastomamultiforme cells; and anti-S1P antibodies significantly reduce theneovascularization associated with animal models of AMD.

The importance of S1P in the angiogenic-dependent tumors makes S1P anexcellent target for cancer treatment. Indeed, antibody neutralizationof extracellular S1P may result in a marked decrease in cancerprogression in mammals, including humans, as a result of inhibition ofblood vessel formation with concomitant loss of the nutrients and oxygenneeded to support tumor growth. Thus, anti-S1P antibodies have severalmechanisms of action, including: (1) direct effects on tumor cellgrowth; (2) indirect anti-angiogenic effects on vascular endothelialcells; and (3) the indirect anti-angiogenic effects that prevent therelease and action of other pro-angiogenic growth factors. Accordingly,anti-S1P antibodies can also serve as anti-metastatic therapeutics, inaddition to providing anti-angiogenic therapy.

Control of angiogenesis is a critical component for the treatment ofother angiogenesis-dependent diseases besides cancer, such asage-related macular degeneration, retinopathy of prematurity, diabeticretinopathy, endometriosis, and rheumatoid arthritis (Carmeliet, P.(2005), Nature, vol. Vol. 438(15): 932-6).

Anti-angiogenic therapeutics are also particularly attractive becausethe vascular endothelial cells that are involved in tumor angiogenesisdo not mutate as easily as do cancer cells; consequently, vascularendothelial cells are less likely than cancer cells to gain resistanceto prolonged therapy, making them useful therapeutic targets. S1Pantibodies, and derivatives thereof, will also be useful in treatingother hyperproliferative disorders associated with S1P activity, such asthose cause by aberrant endothelial cell proliferation, as occurs withthe angiogenesis associated with AMD.

iii. Fibrogenesis and Scarring

(a) S1P, Fibroblasts, and the Remodeling Process

It is clear that cardiac fibroblasts, particularly myofibroblasts, arekey cellular elements in scar formation in response to the cell deathand inflammation of a myocardial infarction (MI). Myofibroblast collagengene expression is a hallmark of remodeling and necessary for scarformation. In addition to its other activities, S1P is also aninflammatory mediator that makes profound contributions to wound healingby activating fibroblast migration and proliferation, in addition toactivating platelets, stimulating angiogenesis, and promoting smoothmuscle function. Thus, S1P, perhaps produced locally by injuredmyocardium, could, in part, be responsible for the maladaptive woundhealing associated with cardiac remodeling and failure, particularly byactivating myofibroblasts in the heart.

There are three general responses of cells to S1P: protection from celldeath; stimulation of proliferation; and the promotion of migratoryresponses. Accordingly, S1P activity or involvement with a particulardisorder, cell line, etc. can be assessed by adapting assays of thissort for this purpose. There is evidence that fibroblasts respond to S1Pin all three ways to promote wound healing. For instance, in several ofthe examples in the Example section below, evidence is presented thatdemonstrates that S1P contributes to remodeling by promoting cardiacmyofibroblast activity (proliferation, migration, and collagen geneexpression).

Anti-S1P antibodies or antibody derivatives will also prevent excessscarring associated with surgical procedures. Excess scarring postinjury or surgery, a problem in adult but not fetal skin tissue (Adzickand Lorenz (1994), Ann Surg, vol. 220: 10-18), is attributed to excessTGF-β in adult skin tissue post injury. S1P has been implicated as apotent activator the TGF-β signaling system. Accordingly, an antiS1Pantibody would be expected to limit excess scarring post injury orsurgery.

(b) Protection from Cell Death by LPA and S1P

LPA is an agent that protects cancer cells from apoptosis. Thus, asdiscussed in detail above, an antibody to LPA, for example, will makecancer cells more susceptible to chemotherapy. This has, in fact, beendemonstrated in the examples hereinbelow, using newly developed anti-LPAmonoclonal antibodies.

As is the case for many cell types, fibroblasts are directly protectedfrom apoptosis by addition of S1P, and apoptosis is enhanced byinhibitors of SPHK, and S1P blocks cytochrome C release and theresultant caspase activation. Further, fibroblasts transfected withSPHK1 exhibit protection from apoptosis, an effect that may depend upontranslocation of SPHK1 to the plasma membrane. It is well-establishedthat SPHK1 up-regulates Akt, thereby regulating Bcl-2 family members andprotecting from apoptosis. Also, S1P₃ is required for Aktphosphorylation in mouse embryonic fibroblasts (MEFs). Also,up-regulation of SPHK and resulting increases in S1P levels protectcardiofibroblasts from apoptosis.

Ceramide, an upstream metabolite of S1P, decreases mitochondrialmembrane potential coincident with increasing the transcription of deathinducing mitochondrial proteins. Because of the rheostat mechanism, S1Pmay have the opposite effect and protect cardiac myofibroblasts (i.e.,fully differentiated fibroblasts in the heart) from apoptosis. Indeed,S1P may even activate autophagy as a protection mechanism. These effectscould be reversed by the neutralizing anti-S1P antibodies (or othermolecules that bind and act to sequester S1P).

B. Pain

Bioactive lipids are believed to play important roles in thepathogenesis of pain, including neuropathic pain and pain associatedwith chemotherapy.

The significant role of LPA signaling in the development of neuropathicpain was established using various pharmacological and geneticapproaches, including the use of mice lacking the LPA1 receptor (see.e.g., Ueda, et al. (2006), Pharmacol Ther, vol. 109: 57-77; Inoue, etal. (2004), Nat. Med., vol. 10: 712-8). Wild-type animals with nerveinjury develop behavioral allodynia and hyperalgesia paralleled bydemyelination in the dorsal root and increased expression of both theprotein kinase C isoform within the spinal cord dorsal horn and the 21calcium channel subunit in dorsal root ganglia. Intrathecal injection ofLPA induced behavioral, morphological and biochemical changes similar tothose observed after nerve ligation. In contrast, mice lacking a singleLPA receptor (LPA-1, also known as EDG-2) that activates the Rho-Rhokinase pathway do not develop signs of neuropathic pain after peripheralnerve injury. Inhibitors of Rho and Rho kinase also prevented thesesigns of neuropathic pain. These results imply that receptor-mediatedLPA signaling is crucial in the initiation of neuropathic pain and thatan antibody to LPA would likely alleviate neuropathic pain inindividuals suffering this condition [Moulin, Del. (2006), Pain ResManag, vol. 11, Suppl A: 30A-6A].

In the context of other pain, that associated with chemotherapy is amajor dose limiting toxicity of many small molecule chemotherapeuticagents. Indeed, many cases of chemotherapy-induced pain have beenreported. For instance, Paclitaxel (Taxol), an anti-neoplastic agentderived from the Pacific yew tree Taxus brevifolia), is used to treat avariety of cancers, including ovarian, breast, and non-small cell lungcancer. Paclitaxel's effectiveness, however, is limited by the highlyincidental development of severe painful peripheral neuropathy such asnumbness and burning pain. A monoclonal antibody against a bioactivelipid correlated with such pain, for example, LPA (or a derivative ofsuch an antibody that contains a lipid-binding portion thereof), couldbe administered in combination with Paclitaxel in order to reduce thepain associated with the chemotherapeutic agent. As a result ofameliorating this dose-limiting toxicity, the amount of Paclitaxel to beadministered could be even higher (and thus even more effective) whenused in combination with such a monoclonal antibody or antibodyderivative. In some embodiments, the chemotherapeutic agent (or otherdrug) could be conjugated to or otherwise associated with the antibodyor antibody derivative, for example, by covalently linking the smallmolecule chemotherapeutic agent to the antibody, by linking the smallmolecule chemotherapeutic to a multivalent scaffold to which is alsolinked a monoclonal antibody or at least one bioactive lipid bindingdomain derived from a monoclonal antibody specifically reactive againstthe target bioactive lipid, etc.

C Cardiovascular Diseases and Disorders

Ischemic heart disease is the leading cause of death in the U.S. Eachyear approximately 1.5 million people suffer heart attacks (myocardialinfarctions), of which about one-third (i.e., about 500,000) are fatal.In addition, about 6.75 million Americans suffer from angina pectoris,the most common manifestation of cardiac ischemia. In total, there aremore than 13 million patients living with ischemic heart disease in theU.S. alone. “Ischemia” is a condition associated with an inadequate flowof oxygenated blood to a part of the body, typically caused by theconstriction or blockage of the blood vessels supplying it. Ischemiaoccurs any time that blood flow to a tissue is reduced below a criticallevel. This reduction in blood flow can result from: (i) the blockage ofa vessel by an embolus (blood clot); (ii) the blockage of a vessel dueto atherosclerosis; (iii) the breakage of a blood vessel (a bleedingstroke); (iv) the blockage of a blood vessel due to acutevasoconstriction; (v) a myocardial infarction (when the heart stops, theflow of blood to organs is reduced, and ischemia results); (vi) trauma;(vii) surgery, during which blood flow to a tissue or organ needs to bereduced or stopped to achieve the aims of surgery (e.g., angioplasty,heart and lung/heart transplants); (viii) exposure to certain agents,e.g., dobutamine or adenosine (Lagerqvist, et al. (1992), Br. Heart J.,vol. 68:282-285); or (ix) anti-neoplastic and other chemotherapeuticagents, such as doxorubicin, that are cardiotoxic.

Even if the flow rate (volume/time) of blood is adequate, ischemia maynonetheless occur due to hypoxia, i.e., a condition in which the oxygencontent of blood is insufficient to satisfy normal cellular oxygenrequirements of the affected area(s). Hypoxic blood is, by definition,distinct from normoxic blood, i.e., blood in which the oxygen content issufficient to satisfy normal cellular oxygen requirements. Hypoxicconditions may result from, but are not limited to, forms of heartfailure that adversely affect cardiac pumping such as hypertension,arrhythmias, septic shock, trauma, cardiomyopathies, and congestiveheart disease.

Myocardial ischemic disorders occur when cardiac blood flow isrestricted (ischemia) and/or when oxygen supply to the heart muscle iscompromised (hypoxia) such that the heart's demand for oxygen is not metby the supply. Coronary artery disease (CAD) arising fromarteriosclerosis, particularly atherosclerosis, is the most common causeof ischemia, and has symptoms such as stable or unstable anginapectoris. CAD can lead to acute myocardial infarctions (AMI) and suddencardiac death. The spectrum of ischemic conditions that results in heartfailure is referred to as Acute Coronary Syndrome (ACS). Reperfusioninjury is often a consequence of ischemia, in particular whenanti-coagulants, thrombolytic agents, or anti-anginal medications areused or when the cardiac vasculature is surgically opened by angioplastyor by coronary artery grafting.

Presently, treatments for acute myocardial infarction and other cardiacdiseases include, but are not limited, to mechanical devices andassociated procedures therewith, e.g., coronary angioplasty;thrombolytic agents such as streptokinase, tPA, and derivatives thereof.Adjuvants to these therapies include beta-blockers, aspirin and heparin,and glycoprotein (GP) IIb/IIIa inhibitors. GP IIb/IIIa inhibitorsdecrease platelet aggregation and thrombus formation. Examples includemonoclonal antibodies (e.g., abciximab), cyclic peptides (e.g.,eptifibatide), and nonpeptide peptidomimetics (e.g., tirofibian,lamifiban, xemilofiban, sibrafiban, and lefradafibian).

Preventive treatments include those that reduce a patient's cholesterollevels by, e.g., diet management and pharmacological intervention.Statins are one type of agent used to reduce cholesterol levels. Statinsare believed to act by inhibiting the activity of HMG-CoA reductase,which in turn increases the hepatic production of cholesterol receptors.Hepatic cholesterol receptors bind cholesterol and remove it from blood.Such agents include lovastatin, simvastatin, pravastatin, andfluvastatin. These and other statins slow the progression of coronaryartery disease, and may induce regression of atherosclerotic lesions inpatients, although the range of side effects from the use of such drugsis not fully understood.

As will be appreciated, monoclonal antibodies and derivatives, and otherfragments and variants reactive against a bioactive lipid may be used toeffect cardiac therapy, alone or in combination with other therapeuticapproaches, including treatment with drugs and/or surgery. Here,“cardiac therapy” refers to the prevention and/or treatment ofmyocardial diseases, disorders, or physical trauma, including myocardialischemia, AMI, CAD, and ACS, as well as trauma or cardiac cell andtissue damage that may occur during or as a consequence ofinterventional cardiology or other surgical or medical procedures ortherapies that may cause ischemic or ischemic/reperfusion damage inmammals, particularly humans.

Besides the heart and brain, an anti-S1P approach can also be applied toother vascular-based, stroke-like conditions such as various inner earpathologies (Scherer, et al. (2006), Cardiovasc Res, vol. 70:79-87).

D. Cerebrovascular Diseases and Disorders

Patients experiencing cerebral ischemia often suffer from disabilitiesranging from transient neurological deficit to irreversible damage(stroke) or death. Cerebral ischemia, i.e., reduction or cessation ofblood flow to the central nervous system, can be characterized as eitherfocal or global. Focal cerebral ischemia refers to cessation orreduction of blood flow within the cerebral vasculature resulting from apartial or complete occlusion in the intracranial or extracranialcerebral arteries. Such occlusion typically results in stroke, asyndrome characterized by the acute onset of a neurological deficit thatpersists for at least 24 hours, reflecting focal involvement of thecentral nervous system and is the result of a disturbance of thecerebral circulation. Other causes of focal cerebral ischemia includevasospasm due to subarachnoid hemorrhage or iatrogenic intervention.

Global cerebral ischemia refers to reduction of blood flow within thecerebral vasculature resulting from systemic circulatory failure, whichpromptly leads to a reduction in oxygen and nutrients to tissues. Thus,global cerebral ischemia results from severe depression of cardiacperformance, and is most frequently caused by AMI, although bothercauses include pump failure resulting from acute myocarditis ordepression of myocardial contractility following cardiac arrest orprolonged cardiopulmonary bypass; mechanical abnormalities, such assevere valvular stenosis, massive aortic or mitral regurgitation, andacutely acquired ventricular septal defects; as well as from cardiacarrhythmia, such as ventricular fibrillation, or from interventionalprocedures, such as carotid angioplasty, stenting, endarterectomy,cardiac catheterization, electrophysiologic studies, and angioplasty.

Ischemic injury post stroke and/or MI typically leads to cell death bydepolarization of critical cells with resulting rise in intracellularNa⁺ and Ca⁺⁺ followed by cell death. One channel controlling thisprocess is the Transient Receptor Potential Protein, a non-voltagedependent channel and recently S1P was identified as an activator ofthis channel through a GPCR-dependent mechanism. In addition, TransientReceptor Potential Protein, sphingosine kinase 1 and sphingokinase 2share promoter regions with Egr-1, an important master switch believedto regulate cardiovascular pathobiology (Khachigian, L M (2006), CircRes, vol. 98: 186-91) and Sp1, a transcription factor that plays acritical role in the death of neural cells (Simard, et al. (2006), NatMed., vol. 12: 433-40). Based on these findings, an antibody to S1Pwould be expected to mitigate cell death caused by ischemia posthypoxia.

Those skilled in the art are easily able to identify patients having astroke or at risk of having a stroke, cerebral ischemia, head trauma, orepilepsy. For example, patients who are at risk of having a strokeinclude those having hypertension or undergoing major surgery.Traditionally, emergent management of acute ischemic stroke consists ofmainly general supportive care, e.g. hydration, monitoring neurologicalstatus, blood pressure control, and/or anti-platelet or anti-coagulationtherapy. Heparin has been administered to stroke patients with limitedand inconsistent effectiveness. In some circumstances, the ischemiaresolves itself over a period of time due to the fact that some thrombiget absorbed into the circulation, or fragment and travel distally overa period of a few days. Tissue plasminogen activator (t-PA) or has beenapproved for treating acute stroke, although such systemic treatment hasbeen associated with increased risk of intracerebral hemorrhage andother hemorrhagic complications. Aside from the administration ofthrombolytic agents and heparin, there are no therapeutic optionscurrently on the market for patients suffering from occlusion focalcerebral ischemia. Vasospasm may be partially responsive to vasodilatingagents. The newly developing field of neurovascular surgery, whichinvolves placing minimally invasive devices within the carotid arteriesto physically remove the offending lesion, may provide a therapeuticoption for these patients in the future, although this kind ofmanipulation may lead to vasospasm itself.

As will be appreciated, antibodies, antibody-derivatives, and otherimmune-derived moiety reactive against a bioactive lipid may be used toeffect cerebrovascular therapy, alone or in combination with othertherapeutic approaches, including treatment with drugs and/or surgery.Here, “cerebrovascular therapy” refers to therapy directed to theprevention and/or treatment of diseases and disorders associated withcerebral ischemia and/or hypoxia. Of particular interest is cerebralischemia and/or hypoxia resulting from global ischemia resulting from aheart disease, as well as trauma or surgical or medical procedures ortherapies that may cause ischemic or ischemic/reperfusioncerebrovascular damage in mammals, particularly humans.

E. Diagnostic and Theranostic Applications for Antibodies that BindBioactive Lipids

As the role of various bioactive lipids in disease is elucidated, newroles for antibody binders of bioactive lipids in diagnostics andtheranostics may also be envisioned. According to the instant invention,methods are provided for enhanced detection of bioactive lipids usingderivatized lipids bound to a solid support. In addition to use of thesedetection methods in antibody production and characterization and inresearch, enhanced detection of bioactive lipids may also provide avaluable diagnostic approach for diseases associated with bioactivelipids. When combined with other techniques, a theranostic approach fordesigning optimal patient treatment is provided. One nonlimiting exampleis use of anti-S1P antibodies in diagnostic and theranostic methodsrelating to the role of S1P as a biomarker for cancer. Diagnostic andtheranostic methods using antibodies targeted to LPA or other bioactivelipids, and for other disease indications, are also envisioned.

Recently, scientific literature has suggested that S1P is a potenttumorigenic growth factor that is likely released from tumor cells, andthat S1P may be a novel biomarker for early-stage cancer detection.SPHK, the enzyme which is responsible for the production of S1P, issignificantly up-regulated in a variety of cancer types (French,Schrecengost et al. 2003). SPHK activity is up-regulated 2-3 fold inmalignant breast, colon, lung, ovarian, stomach, uterine, kidney andrectal cancer when compared to adjacent normal tissue. These workersalso showed that SPHK expression varies from patient to patient,suggesting that the tumors of some patients might be more dependent onS1P than those of other patients with the same tumor type. Searchingcommercially available genomics database (ASCENTA, Genelogic Inc.,Gaithersburg Md.) confirms that the relative expression of SPHK is, ingeneral, significantly elevated in a wide variety of malignant tumors.

Recent publications have also suggested that S1P may be a novel cancerbiomarker [Xu, Y. et al., (1998) JAMA 280: 719-723; Shen, Z. et al.,(2001) Gynecol Oncol 83: 25-30; Xiao, Y. J. et al., (2001) Anal Biochem290(2): 302-13; Sutphen (2004) Cancer Epidemiology 13(7) 1185-91]. Forexample, Sutphen et al, have shown that serum S1P levels are elevated inearly-stage ovarian cancer patients (Sutphen 2004). One might predictfrom the data that breast cancer patients might also demonstrate somevariability in their dependence on S1P. Taken together, thesepreliminary observations suggest that the success of an anti-S1Ptherapeutic, e.g., an anti-S1P mAb therapeutic, might be predicted foran individual patient if that patient's biopsy tissue, blood, urine orother tissue or fluid sample show elevated S1P levels.

The potential use of S1P in biological fluids has been disclosed in thefollowing patents, all of which are commonly assigned with the instantapplication. U.S. Pat. No. 6,534,323, U.S. Pat. No. 6,534,322; U.S. Pat.No. 6,210,976; U.S. Pat. No. 6,858,383; U.S. Pat. No. 6,881,546; U.S.Pat. No. 7,169,390 and U.S. Pat. No. 6,500,633.

Even though humanized antibodies have low toxicity and large therapeuticindices, they are quite costly to the patient and to health careproviders. Thus directing utility of the anti-S1P mAb therapeutic tothose who would most likely respond to this treatment would lower risksand minimize costs, while providing optimum patient benefit.

Outlined below are a few proposed applications of biolipid diagnosticsand theranostics for improved disease management.

1. S1P may be used as a biomarker to predict individual patienttherapeutic efficacy especially when combined with sphingolipid-basedgenomics. Based on recent findings, we would predict that S1P dependenttumors may produce their own S1P in addition to the abundant serumsource of S1P. Highly aggressive tumors utilize a strategy of producingtheir own growth factors, and we suggest that S1P is one of the growthfactors. Therefore, serum, plasma or urine measurements of total S1Pfrom individual patients would be one predictor of patient outcome.Moreover, S1P production would be concentrated in the tumor itself andin the tumor microenvironment (e.g., interstitial fluid). Example 11hereinbelow describes the use of an anti-S1P mAb in animmunohistochemical method of a tumor section to assess S1P productionby the tumor itself. Up-regulation of SPHK may prove useful, but sincethe kinase is an enzyme, it is believed that the signal as measured byS1P production will be much higher than if one relied on RNA or proteinexpression of the kinase itself. In addition, it is hypothesized thatpatients whose tumors have an up-regulation of S1P-receptors and SPHKexpression are more likely to have tumors that rely on S1P as a growthfactor. It is believed that these patients would benefit most from ourputative anti-S1P mAb therapy. Therefore, bioassays from biopsy tissueanalyzed by quantitative-PCR for the relative expression of S1Preceptors and SPHK would provide a strong theranostic platform. Thistheranostic platform would consist of serum S1P marker analysis incombination with the genomic or proteomic quantification of S1P-relatedprotein markers as surrogate markers of disease. This novel multi-markeranalysis would provide a very strong platform for prediction ofindividual responsiveness to an anti-S1P mAb (SPHINGOMAB™)-basedtherapy.

2. S1P may be used as a surrogate marker to titrate therapeutic regimen.The concentration of serum S1P from patients being treated with theanti-S1P mAb has the potential to be used as a surrogate marker forevaluating the course of treatment. An ELISA-based platform usingpatient serum, plasma or urine samples will allow for the accuratemeasurement of the S1P biomarker levels and to determine more preciselythe anti-S1P mAb dosing regimen for individuals. Surrogate marker levelscould be used in combination with the standard clinical endpoints todetermine efficacy of the medical regimen.

3. S1P may be used as a screening tool for the early detection ofcancer. The early detection of cancer is of concern due to the strongcorrespondence of stage of progression and success of therapy. Stage Iof ovarian cancer is very difficult to detect due to the fact thatmajority of patients are asymptomatic. By the time ovarian cancer isdiagnosed, most patients are in the later stages of the disease.Detection at an earlier stage has obvious benefits to patient outcome.As described above, ovarian cancer patient serum contains a 2-foldelevation of S1P, and this elevation is easily detectable with ourcurrent ELISA platform. Since many solid tumor types, including ovariancancer, exhibit elevated SPHK expression, it is presumed that many ofthe patients with these cancers would display elevated blood and/orurine S1P that could allow the clinician to intervene earlier in diseaseprogression.

Derivatized bioactive lipids described herein can also be used to detectthe level of antibodies in a fluid or tissue sample of a patient.Without being limited by the following, such immunoassays that detectthe presence of anti-sphingolipid antibodies in blood and can be used toindirectly test for increased sphingolipids in patients with chronicischemic conditions, cancer or autoimmune disorders such as multiplesclerosis. This assay is based on the assumption that patients produceanti-sphingolipid antibodies as a consequence of elevated blood levelsof sphingolipids by analogy to the anti-lactosylsphingosine antibodiesobserved in patients with colorectal cancer (Jozwiak W. & J. Koscielak,Eur. J. Cancer Clin. Oncol. 18:617-621, 1982) and theanti-galactocerebroside antibodies detected in the sera of leprosypatients (Vemuri N. et al., Leprosy Rev. 67:95-103, 1996).

F. Research

The bioactive signaling lipid targets of the invention are readilyadaptable for use in high-throughput screening assays for screeningcandidate compounds to identify those which have a desired activity,e.g., inhibiting an enzyme that catalyzes a reaction that produces anundesirable bioactive signaling lipid, or blocking the binding of abioactive signaling lipid to a receptor therefore. The compounds thusidentified can serve as conventional “lead compounds” or can themselvesbe used as therapeutic agents. The methods of screening of the inventioncomprise using screening assays to identify, from a library of diversemolecules, one or more compounds having a desired activity. A “screeningassay” is a selective assay designed to identify, isolate, and/ordetermine the structure of, compounds within a collection that have apre-selected activity. The collection can be a traditional combinatoriallibraries are prepared according to methods known in the art, or may bepurchased commercially and may be a wide-range of organic structures orstructures pre-selected for potential bioactive signaling activity. By“identifying” it is meant that a compound having a desirable activity isisolated, its chemical structure is determined (including withoutlimitation determining the nucleotide and amino acid sequences ofnucleic acids and polypeptides, respectively) the structure of, and,additionally or alternatively, purifying compounds having the screenedactivity. Biochemical and biological assays are designed to test foractivity in a broad range of systems ranging from protein-proteininteractions, enzyme catalysis, small molecule-protein binding, tocellular functions. Such assays include automated, semi-automated assaysand high throughput screening assays.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These Examples are in no way to be considered tolimit the scope of the invention in any manner.

Example 1 Synthetic Scheme for Making a Representative Thiolated Analogof S1P

The synthetic approach described in this example results in thepreparation of an antigen by serial addition of structural elementsusing primarily conventional organic chemistry. A scheme for theapproach described in this example is provided in FIG. 1, and thecompound numbers in the synthetic description below refer to thenumbered structures in FIG. 1.

This synthetic approach began with the commercially available15-hydroxyl pentadecyne, 1, and activation by methyl sulphonyl chlorideof the 15-hydroxy group to facilitate hydroxyl substitution to producethe sulphonate, 2. Substitution of the sulphonate with t-butyl thiolyielded the protected thioether, 3, which was condensed with Garner'saldehyde to produce 4. Gentle reduction of the alkyne moiety to analkene (5), followed by acid catalyzed opening of the oxazolidene ringyielded S-protected and N-protected thiol substituted sphingosine, 6.During this last step, re-derivatization with di-t-butyl dicarbonate wasemployed to mitigate loss of the N-BOC group during the acid-catalyzedring opening.

As will be appreciated, compound 6 can itself be used as an antigen forpreparing haptens to raise antibodies to sphingosine, or, alternatively,as starting material for two different synthetic approaches to prepare athiolated S1P analog. In one approach, compound 6 phosphorylation withtrimethyl phosphate produced compound 7. Treatment of compound 7 withtrimethylsilyl bromide removed both methyl groups from the phosphate andthe t-butyloxycarbonyl group from the primary amine, leaving compound 8with the t-butyl group on the sulfur as the only protecting group. Toremove this group, the t-butyl group was displaced by NBS to form thedisulfide, 9, which was then reduced to form the thiolated S1P analog,10.

Another approach involved treating compound 6 directly with NBSCl toform the disulfide, 11, which was then reduced to form the N-protectedthiolated S1P analog, 12. Treatment of this compound with mild acidyielded the thiolated sphingosine analog, 13, which can bephosphorylated enzymatically with, e.g., sphingosine kinase, to yieldthe thiolated S1P analog, 10.

Modifications of the presented synthetic approach are possible,particularly with regard to the selection of protecting andde-protecting reagents, e.g., the use of trimethyl disulfide triflatedescribed in Example 3 to de-protect the thiol.

Compound 2.

DCM (400 mL) was added to a 500 mL RB flask charged with 1 (10.3 g,45.89 mmol), and the resulting solution cooled to 0° C. Next, TEA (8.34g, 82.60 mmol, 9.5 mL) was added all at once followed by MsCl (7.88 g,68.84 mmol, 5.3 mL) added drop wise over 10 min. The reaction wasallowed to stir at RT for 0.5 h or until the disappearance of startingmaterial (R_(f)=0.65, 5:1 hexanes:EtOAc). The reaction was quenched withNH₄Cl (300 mL) and extracted (2×200 mL) DCM. The organic layers weredried over MgSO₄, filtered and the filtrate evaporated to a solid (13.86g, 99.8% yield). ¹H NMR (CDCl₃) δ 4.20 (t, J=6.5 Hz, 2H), 2.98 (s, 3H),2.59 (td, J=7 Hz, 3 Hz, 2H), 1.917 (t, J=3 Hz, 1H), 1.72 (quintet, J=7.5Hz, 2H), 1.505 (quintet, J=7.5 Hz, 2H), 1.37 (br s, 4H), 1.27 (br s,14H). ¹³C{¹H} NMR (CDCl₃) δ 85.45, 70.90, 68.72, 46.69, 38.04, 30.22,30.15, 30.14, 30.07, 29.81, 29.76, 29.69, 29.42, 29.17, 26.09, 19.06,9.31. The principal ion observed in a HRMS analysis (ES-TOF) of compound2 was m/z=325.1804 (calculated for C₁₆H₃₀O₃S: M+Na⁺ 325.1808).

Compound 3.

A three-neck IL RB flask was charged with t-butylthiol (4.54 g, 50.40mmol) and THF (200 mL) and then placed into an ice bath. n-BuLi (31.5 mLof 1.6 M in hexanes) was added over 30 min. Next, compound 2 (13.86 g,45.82 mmol), dissolved in THF (100 mL), was added over 2 min. Thereaction is allowed to stir for 1 hour or until starting materialdisappeared (R_(f)=0.7, 1:1 hexanes/EtOAc). The reaction was quenchedwith saturated NH₄Cl (500 mL) and extracted with EtO₂ (2×250 mL), driedover MgSO₄, filtered, and the filtrate evaporated to yield a yellow oil(11.67 g, 86% yield). ¹H NMR (CDCl₃) δ 2.52 (t, J=7.5 Hz, 2H), 2.18 (td,J=7 Hz, 2.5 Hz, 2H), 1.93 (t, J=2.5 Hz, 1H), 1.55 (quintet, J=7.5 Hz,2H), 1.51 (quintet, J=7 Hz, 2H), 1.38 (br s, 4H), 1.33 (s, 9H), 1.26 (s,14H). ¹³C{¹H} NMR (CDCl₃) δ 85.42, 68.71, 68.67, 54.07, 42.37, 31.68,30.58, 30.28, 30.26, 30.19, 30.17, 29.98, 29.78, 29.44, 29.19, 29.02,19.08.

Compound 4.

A 250 mL Schlenk flask charged with compound 3 (5.0 g, 16.85 mmol) wasevacuated and filled with nitrogen three times before dry THF (150 mL)was added. The resulting solution cooled to −78° C. Next, n-BuLi (10.5mL of 1.6M in hexanes) was added over 2 min. and the reaction mixturewas stirred for 18 min. at −78° C. before the cooling bath was removedfor 20 min. The dry ice bath was returned. After 15 min., Garner'saldehyde (3.36 g, 14.65 mmol) in dry THF (10 mL) was then added over 5min. After 20 min., the cooling bath was removed. Thin layerchromatography (TLC) after 2.7 hr. showed that the Garner's aldehyde wasgone. The reaction was quenched with saturated aqueous NH₄Cl (300 mL)and extracted with Et₂O (2×250 mL). The combined Et₂O phases were driedover Na₂SO₄, filtered, and the filtrate evaporated to give crudecompound 4 and its syn diastereomer (not shown in FIG. 1) as a yellowoil (9.06 g). This material was then used in the next step withoutfurther purification.

Compound 5.

To reduce the triple bond in compound 4, the oil was dissolved in dryEt₂O (100 mL) under nitrogen. RED-Al (20 mL, 65% in toluene) was slowlyadded to the resulting solution at RT to control the evolution ofhydrogen gas (H₂). The reaction was allowed to stir at RT overnight orwhen TLC showed the disappearance of the starting material (R_(f)=0.6 in1:1 EtOAc:hexanes) and quenched slowly with cold MeOH or aqueous NH₄Clto control the evolution of H₂. The resulting white suspension wasfiltered through a Celite pad and the filtrate was extracted with EtOAc(2×400 mL). Combined EtOAc extracts were dried over MgSO₄, filtered, andthe filtrate evaporated to leave crude compound 5 and its syndiastereomer (not shown in FIG. 1) as a yellow oil (7.59 g).

Compound 6.

The oil containing compound 5 was dissolved in MeOH (200 mL), PTSAhydrate (0.63 g) was added, and the solution stirred at RT for 1 day andthen at 50° C. for 2 days, at which point TLC suggested that allstarting material (5) was gone. However, some polar material waspresent, suggesting that the acid had partially cleaved the BOC group.The reaction was worked up by adding saturated aqueous NH₄Cl (400 mL),and extracted with ether (3×300 mL). The combined ether phases weredried over Na₂SO₄, filtered, and the filtrate evaporated to dryness,leaving 5.14 g of oil. In order to re-protect whatever amine had formed,the crude product was dissolved in CH₂Cl₂ (150 mL), to which was addedBOC₂O (2.44 g) and TEA (1.7 g). When TLC (1:1 hexanes/EtOAc) showed nomore material remaining on the baseline, saturated aqueous NH₄Cl (200mL) was added, and, after separating the organic phase, the mixture wasextracted with CH₂Cl₂ (3×200 mL). Combined extracts were dried overNa₂SO₄, filtered, and the filtrated concentrated to dryness to yield ayellow oil (7.7 g) which was chromatographed on a silica column using agradient of hexanes/EtOAc (up to 1:1) to separate the diastereomers. ByTLC using 1:1 PE/EtOAc, the R_(f) for the anti isomer, compound 6, was0.45. For the syn isomer (not shown in FIG. 1) the R_(f) was 0.40. Theyield of compound 6 was 2.45 g (39% overall based on Garner's aldehyde).¹H NMR of anti isomer (CDCl₃) δ 1.26 (br s, 20H), 1.32 (s, 9H), 1.45 (s,9H), 1.56 (quintet, 2H, J=8 Hz), 2.06 (q, 2H, J=7 Hz), 2.52 (t, 2H, J=7Hz), 2.55 (br s, 2H), 3.60 (br s, 1H), 3.72 (ddd, 1H, J=11.5 Hz, 7.0 Hz,3.5 Hz), 3.94 (dt, 1H, J=11.5 Hz, 3.5 Hz), 4.32 (d, 1H, J=4.5 Hz), 5.28(br s, 1H), 5.54 (dd, 1H, J=15.5 Hz, 6.5 Hz), 5.78 (dt, 1H, J=15.5 Hz,6.5 Hz). ¹³C {¹H} NMR (CDCl₃) δ 156.95, 134.80, 129.66, 80.47, 75.46,63.33, 56.17, 42.44, 32.98, 31.70, 30.58, 30.32, 30.31, 30.28, 30.20,30.16, 30.00, 29.89, 29.80, 29.08, 29.03.

Anal. Calculated for C₂₇H₅₃NO₄S: C, 66.48; H, 10.95; N, 2.87. Found: C,65.98; H, 10.46; N, 2.48.

Compound 7.

To a solution of the alcohol compound 6 (609.5 mg, 1.25 mmol) dissolvedin dry pyridine (2 mL) was added CBr₄ (647.2 mg, 1.95 mmol, 1.56 equiv).The flask was cooled in an ice bath and P(OMe)₃ (284.7 mg, 2.29 mmol,1.84 equiv) was added drop wise over 2 min. After 4 min. the ice bathwas removed and after 12 hr. the mixture was diluted with ether (20 mL).The resulting mixture washed with aqueous HCl (10 mL, 2 N) to form anemulsion which separated on dilution with water (20 mL). The aqueousphase was extracted with ether (2×10 mL), then EtOAc (2×10 mL). Theether extracts and first EtOAc extract were combined and washed withaqueous HCl (10 mL, 2 N), water (10 mL), and saturated aqueous NaHCO₃(10 mL). The last EtOAc extract was used to back-extract the aqueouswashes. Combined organic phases were dried over MgSO₄, filtered, and thefiltrate concentrated to leave crude product (1.16 g), which waspurified by flash chromatography over silica (3×22 cm column) usingCH₂Cl₂, then CH₂Cl₂-EtOAc (1:20, 1:6, 1:3, and 1:1—product started toelute, 6:4, 7:3). Early fractions contained 56.9 mg of oil. Laterfractions provided product (compound 7, 476.6 mg, 64%) as clear,colorless oil.

Anal. Calculated for C₂₉H₅₈NO₇PS (595.82): C, 58.46; H, 9.81; N, 2.35.Found: C, 58.09; H, 9.69; N, 2.41.

Compound 8.

A flask containing compound 7 (333.0 mg, 0.559 mmol) and a stir bar wasevacuated and filled with nitrogen. Acetonitrile (4 mL, distilled fromCaH₂) was injected by syringe and the flask now containing a solutionwas cooled in an ice bath. Using a syringe, (CH₃)₃SiBr (438.7 mg, 2.87mmol, 5.13 equiv.) was added over the course of 1 min. After 35 min.,the upper part of the flask was rinsed with an additional portion ofacetonitrile (1 mL) and the ice bath was removed. After another 80 min.,an aliquot was removed, the solution dried by blowing nitrogen gas overit, and the residue analyzed by ¹H NMR in CDCl₃, which showed onlytraces of peaks ascribed to P—OCH₃ moieties. After 20 min., water (0.2mL) was added to the reaction mixture, followed by the CDCl₃ solutionused to analyze the aliquot, and the mixture was concentrated to ca. 0.5mL volume on a rotary evaporator. Using acetone (3 mL) in portions theresidue was transferred to a tared test tube, forming a pale brownsolution. Water (3 mL) was added in portions. After addition of 0.3 mL,cloudiness was seen. After a total of 1 mL, a gummy precipitate hadformed. As an additional 0.6 mL of water was added, more cloudiness andgum separated, but the final portion of water seemed not to change theappearance of the mixture. Overall, this process was accomplished over aperiod of several hours. The tube was centrifuged and the supernatantremoved by pipet. The solid, no longer gummy, was dried over P₄O₁₀ invacuo, leaving compound 8 (258.2 mg, 95%) as a monohydrate.

Anal. Calculated. for C₂₂H₄₆NO₅PS+H₂O (485.66): C, 54.40; H, 9.96; N,2.88. Found: C, 54.59; H, 9.84; N, 2.95.

Compound 9.

Compound 8 (202.6 mg, 0.417 mmol) was added in a glove box to a testtube containing a stir bar, dry THF (3 mL) and glacial HOAc (3 mL).NBSCl (90 mg, 0.475 mmol, 1.14 equiv) were added, and after 0.5 hr., aclear solution was obtained. After a total of 9 hr., an aliquot wasevaporated to dryness and the residue analyzed by ¹H NMR in CDCl₃. Thepeaks corresponding to CH₂StBu and CH₂SSAr suggested that reaction wasabout 75% complete, and comparison of the spectrum with that of pureNBSCl in CDCl₃ suggested that none of the reagent remained in thereaction. Therefore, an additional portion (24.7 mg, 0.130 mmol, 0.31equiv) was added, followed 3 hr. later by an additional portion (19.5mg, 0.103 mmol, 0.25 equiv). After another 1 hr., the mixture wastransferred to a new test tube using THF (2 mL) to rinse and water (1mL) was added.

Compound 10.

PMe₃ (82.4 mg, 1.08 mmol, 1.52 times the total amount of2-nitrobenzenesulfenyl chloride added) was added to the clear solutioncompound 9 described above. The mixture grew warm and cloudy, withprecipitate forming over time. After 4.5 hr., methanol was added, andthe tube centrifuged. The precipitate settled with difficulty, occupyingthe bottom 1 cm of the tube. The clear yellow supernatant was removedusing a pipet. Methanol (5 mL, deoxygenated with nitrogen) was added,the tube was centrifuged, and the supernatant removed by pipet. Thiscycle was repeated three times. When concentrated, the final methanolwash left only 4.4 mg of residue. The bulk solid residue was dried overP₄O₁₀ in vacuo, leaving compound 10 (118.2 mg, 68%) as amonohydrochloride.

Anal. Calculated for C₁₈H₃₈NO₅S+HCl (417.03): C, 51.84; H, 9.43; N,3.36. Found: C, 52.11; H, 9.12; N, 3.30.

Compound 11.

Compound 6 (1.45 g, 2.97 mmol) was dissolved in AcOH (20 mL), and NBSCl(0.56 g, 2.97 mmol) was added all at once. The reaction was allowed tostir for 3 hr. or until the disappearance of the starting material andappearance of the product was observed by TLC [product R_(f)=0.65,starting material R_(f)=0.45, 1:1 EtOAc/hexanes]. The reaction wasconcentrated to dryness on a high vacuum line and the residue dissolvedin THF/H₂O (100 mL of 10:1).

Compound 12.

Ph₃P (0.2.33 g, 8.91 mmol) was added all at once to the solution abovethat contained compound 11 and the reaction was allowed to stir for 3hr. or until the starting material disappeared. The crude reactionmixture was concentrated to dryness on a high vacuum line, leaving aresidue that contained compound 12.

Compound 13.

The residue above containing compound 12 was dissolved in DCM (50 mL)and TFA (10 mL). The mixture was stirred at RT for 5 hr. andconcentrated to dryness. The residue was the loaded onto a column withsilica gel and chromatographed with pure DCM, followed by DCM containing5% MeOH, then 10% MeOH, to yield the final product, compound 13, as asticky white solid (0.45 g, 46% yield from 5). ¹H NMR (CDCl₃) δ 1.27(s), 1.33 (br m,), 1.61 (p, 2H, J=7.5 Hz), 2.03 (br d, 2H, J=7 Hz), 2.53(q, 2H, J=7.5 Hz), 3.34 (br s, 1H), 3.87 (br d, 2H, J=12 Hz), 4.48 (brs, 2H), 4.58 (br s, 2H), 5.42 (dd, 1H, J=15 Hz, 5.5 Hz), 5.82 (dt, 1H,J=15 Hz, 5.5 Hz), 7.91 (br s, 4H). ¹³C{¹H} NMR (CDCl₃) δ 136.85, 126.26,57.08, 34.76, 32.95, 30.40, 30.36, 30.34, 30.25, 30.19, 30.05, 29.80,29.62, 29.09, 25.34.

Example 2 Synthetic Schemes for Making Thiolated Fatty Acids

The synthetic approach described in this example details the preparationof a thiolated fatty acid to be incorporated into a more complex lipidstructure that could be further complexed to a protein or other carrierand administered to an animal to elicit an immune response. The approachuses using conventional organic chemistry. A scheme showing the approachtaken in this example is provided in FIG. 2, and the compound numbers inthe synthetic description below refer to the numbered structures in FIG.2.

Two syntheses are described. The first synthesis, for a C-12 thiolatedfatty acid, starts with the commercially available 12-dodecanoic acid,compound 14. The bromine is then displaced with t-butyl thiol to yieldthe protected C-12 thiolated fatty acid, compound 15. The secondsynthesis, for a C-18 thiolated fatty acid, starts with the commerciallyavailable 9-bromo-nonanol (compound 16). The hydroxyl group in compound16 is protected by addition of a dihydropyran group and the resultingcompound, 17, is dimerized through activation of half of the brominatedmaterial via a Grignard reaction, followed by addition of the otherhalf. The 18-hydroxy octadecanol (compound 18) produced followingacid-catalyzed removal of the dihydropyran protecting group isselectively mono-brominated to form compound 19. During this reactionapproximately half of the alcohol groups are activated for nucleophilicsubstitution by formation of a methane sulfonic acid ester. The alcoholis then oxidized to form the 18-bromocarboxylic acid, compound 20, whichis then treated with t-butyl thiol to displace the bromine and form theprotected, thiolated C-18 fatty acid, compound 21.

The protected thiolated fatty acids, each a t-butyl thioether, can beincorporated into a complex lipid and the protecting group removedusing, e.g., one of the de-protecting approaches described in Examples 1and 3. The resulting free thiol then can be used to complex with aprotein or other carrier prior to inoculating animal with the hapten.

A. Synthesis of a C-12 Thiolated Fatty Acid

Compound 15.

t-Butyl thiol (12.93 g, 143 mmol) was added to a dry Schlenk flask, andSchlenk methods were used to put the system under nitrogen. Dry,degassed THF (250 mL) was added and the flask cooled in an ice bath.n-BuLi (55 mL of 2.5 M in hexanes, 137.5 mmol) was slowly added over 10min by syringe. The mixture was allowed to stir at 0° C. for an hour.The bromoacid, compound 14 (10 g, 36 mmol), was added as a solid and thereaction heated and stirred at 60° C. for 24 hr. The reaction wasquenched with 2 M HCl (250 mL), and extracted with ether (2×300 mL). Thecombined ethereal layers were dried with magnesium sulfate, filtered,and the filtrate concentrated by rotary evaporation to yield thethioether acid, compound 15 (10 g, 99% yield) as a beige powder. ¹H NMR(CDCl₃, 500 MHz) δ 1.25-1.35 (br s, 12H), 1.32 (s, 9H), 1.35-1.40 (m,2H), 1.50-1.60 (m, 2H), 1.60-1.65 (m, 2H), 2.35 (t, 2H, J=7.5 Hz), 2.52(t, 2H, J=7.5 Hz). Principal ion in HRMS (ES-TOF) was observed at m/z311.2020, calculated for M+Na⁺ 311.2015.

B. Synthesis of a C-12 Thiolated Fatty Acid

Compound 17.

A dry Schlenk flask was charged with compound 16 (50 g, 224.2 mmol) anddissolved in dry degassed THF (250 mL) distilled fromsodium/benzophenone. The flask was cooled in an ice bath and then PTSA(0.5 g, 2.6 mmol) was added. Dry, degassed DHP (36 g, 42.8 mmol) wasthen added slowly over 5 min. The mixture was allowed to warm up to RTand left to stir overnight and monitored by TLC (10:1 PE:EtOAc) untilthe reaction was deemed done by the complete disappearance of the spotfor the bromoalcohol. TEA (1 g, 10 mmol) was then added to quench thePTSA. The mixture was then washed with cold sodium bicarbonate solutionand extracted with EtOAc (3×250 mL). The organic layers were then driedwith magnesium sulfate and concentrated to yield 68.2 g of crude productwhich was purified by column chromatography (10:1 PE:EtOAc) to yield 60g (99% yield) of a colorless oil. ¹H NMR (CDCl₃, 500 MHz) δ 1.31 (br s,6H), 1.41-1.44 (m, 2H), 1.51-1.62 (obscured multiplets, 6H), 1.69-1.74(m, 1H), 1.855 (quintet, J=7.6 Hz, 2H), 3.41 (t, J=7 Hz, 2H), 3.48-3.52(m, 2H), 3.73 (dt, 2H, J=6.5 Hz), 3.85-3.90 (m, 2H), 4.57 (t, 2H, J=3Hz).

Compound 18.

Magnesium shavings (2.98 g, 125 mmol) were added to a flame-driedSchlenk flask along with a crystal of iodine. Dry THF (200 mL) distilledfrom sodium was then added and the system was degassed using Schlenktechniques. Compound 17 (30 g, 97 mmol) was then slowly added to themagnesium over 10 min. and the solution was placed in an oil bath at 65°C. and allowed to stir overnight. The reaction was deemed complete byTLC by quenching an aliquot with acetone and observing the change in RFin a 10:1 PE:EtOAc mixture. The Grignard solution was then transferredby cannula to a three-necked flask under nitrogen containing additionalcompound 17 (30 g, 97 mmol). The flask containing the resulting mixturewas then cooled to 0° C. in an ice bath and a solution of Li₂CuCl₄ (3 mLof 1 M) was then added via syringe. The reaction mixture turned a verydark blue within a few minutes. This mixture was left to stir overnight.The next morning the reaction was deemed complete by TLC (10:1PE:EtOAc), quenched with a saturated NH₄Cl solution, and then extractedinto ether (3×250 mL). The ether layers were dried with magnesiumsulfate and concentrated to yield crude product (40 g), which wasdissolved in MeOH. Concentrated HCl (0.5 mL) was then added, whichresulted in the formation of a white emulsion, which was left to stirfor 3 hr. The white emulsion was then filtered to yield 16 g (58% yield)of the pure diol, compound 18. ¹H NMR (CDCl₃, 200 MHz) δ 1.26 (br s,24H), 1.41-1.42 (m, 4H), 1.51-1.68 (m, 4H), 3.65 (t, 4H, J=6.5 Hz).

Compound 19.

The symmetrical diol, compound 18 (11 g, 38.5 mmol), was added to a drySchlenk flask under nitrogen, then dry THF (700 mL) distilled fromsodium was added. The system was degassed and the flask put in an icebath. Diisopropylethylamine (6.82 mL, 42.3 mmol) was added via syringe,followed by MsCl (3.96 g, 34.4 mmol) added slowly, and the mixture wasleft to stir for 1 hr. The reaction was quenched with saturated NaH₂PO₄solution (300 mL), and then extracted with EtOAc (3×300 mL). The organiclayers were then combined, dried with MgSO₄, and concentrated to yield14 g of a mixture of the diol, monomesylate, and dimesylate. NMR showeda 1:0.8 mixture of CH₂OH:CH₂OMs protons. The mixture was then dissolvedin dry THF (500 mL), deoxygenated, and to it was added LiBr (3.5 g,40.23 mmol). This mixture was allowed reflux overnight, upon which thereaction was quenched with water (150 mL), and extracted with EtOAc(3×250 mL). The organic layer was then dried with MgSO₄, andconcentrated to yield a mixture of brominated products that were thenpurified by flash chromatography (DCM) to yield compound 19 (3.1 g, 25%yield) as a white powder. ¹H NMR (CDCl₃, 500 MHz) δ 1.26 (br s, 26H),1.38-1.46 (m, 2H), 1.55 (quintet, 2H, J=7.5 Hz), 1.85 (quintet, 2H,J=7.5 Hz), 3.403 (t, 2H, J=6.8 Hz), 3.66 (t. 2H, J=6.8 Hz).

Compound 20.

A round bottom flask was charged with compound 19 (2.01 g, 5.73 mmol)and the solid dissolved in reagent grade acetone (150 mL).Simultaneously, Jones reagent was prepared by dissolving CrO₃ (2.25 g,22 mmol) in H₂SO₄ (4 mL) and then slowly adding 10 mL of cold water andletting the solution stir for 10 min. The cold Jones reagent was thenadded to the round bottom flask slowly over 5 min., after which thesolution stirred for 1 hr. The resulting orange solution turned greenwithin several minutes. The mixture was then quenched with water (150mL) extracted twice in ether (3×150 mL). The ether layers were thendried with magnesium sulfate, and concentrated to yield compound 20(2.08 g, 98% yield) as a white powder. ¹H NMR (CDCl₃, 200 MHz) δ 1.27(br s, 26H), 1.58-1.71 (m, 2H), 1.77-1.97 (m, 2H), 2.36 (t, 2H, J=7.4Hz), 3.42 (t, 2H, J=7 Hz).

Compound 21.

t-Butylthiol (11.32 g, 125 mmol) was added to a dry Schlenk flask anddissolved in dry THF (450 mL) distilled from sodium. The solution wasdeoxygenated by bubbling nitrogen through it before the flask was placedin an ice bath. n-BuLi solution in hexanes (70 mL of 1.6 M) was thenadded slowly via syringe over 10 min. This mixture was allowed to stirfor 1 hr., then compound 20 (5.5 g, 16.2 mmol) was added and thesolution was left to reflux at 60° C. overnight. The next morning analiquot was worked up, analyzed by NMR, and the reaction deemedcomplete. The reaction was quenched with HCl (200 mL of 2 M) andextracted with ether (3×250 mL). The ethereal layers were then driedwith magnesium sulfate, filtered, and the filtrate concentrated to yieldthe product, compound 21, as a white solid (5 g, 90% yield). ¹H NMR(CDCl₃, 200 MHz) δ 1.26 (br s, 26H), 1.32 (br s, 9H), 1.48-1.70 (m, 4H),2.35 (t, 2H, J=7.3 Hz), 2.52 (t, 2H, J=7.3 Hz). ¹³C NMR (CDCl₃, 200 MHz)δ 24.69, 28.35, 29.05, 29.21, 29.28, 29.39, 29.55, 29.89, 31.02 (3C),33.98, 41.75, 179.60.

Example 3 Synthetic Scheme for Making a Thiolated Analog of LPA

The synthetic approach described in this example results in thepreparation of thiolated LPA. The LPA analog can then be furthercomplexed to a carrier, for example, a protein carrier, which can thenbe administered to an animal to elicit an immugenic response to LPA.This approach uses both organic chemistry and enzymatic reactions, thesynthetic scheme for which is provided in FIG. 3. The compound numbersin the synthetic description below refer to the numbered structures inFIG. 3.

The starting materials were compound 15 in Example 2 andenantiomerically pure glycerophoshocholine (compound 22). These twochemicals combined to yield the di-acetylated product, compound 23,using DCC to facilitate the esterification. In one synthetic processvariant, the resulting di-acylated glycerophosphocholine was treatedfirst with phospholipase-A2 to remove the fatty acid at the sn-2position of the glycerol backbone to produce compound 24. This substancewas further treated with another enzyme, phospholipase-D, to remove thecholine and form compound 26. In another synthetic process variant, thephospholipase-D treatment preceded the phospholipase-A2 treatment toyield compound 25, and treatment of compound 25 with phospholipase-Dthen yields compound 26. Both variants lead to the same product, thephosphatidic acid derivative, compound 26. The t-butyl protecting groupin compound 26 is then removed, first using trimethyl disulfide triflateto produce compound 27, followed by a disulfide reduction to produce thedesired LPA derivative, compound 28. As those in the art willappreciate, the nitrobenzyl sulfenyl reaction sequence described inExample 1 can also be used to produce compound 28.

Compound 23.

To a flame-dried Schlenk flask were added the thioether acid, compound15 (10 g, 35.8 mmol), compound 22 (glycerophosphocholine-CdCl₂ complex,4.25 g, 8.9 mmol), DCC (7.32 g, 35.8 mmol), and DMAP (2.18 g, 17.8mmol), after which the flask was evacuated and filled with nitrogen. Aminimal amount of dry, degassed DCM was added (100 mL), resulting in acloudy mixture. The flask was covered with foil and then left to stiruntil completed, as by TLC (silica, 10:5:1 DCM:MeOH:concentrated NH₄OH).The insolubility of compound 16 precluded monitoring its disappearanceby TLC, but the reaction was stopped when the product spot of R_(f) 0.1was judged not to be increasing in intensity. This typically required 3to 4 days, and in some cases, addition of more DCC and DMAP. Uponcompletion, the reaction mixture was filtered, and the filtrateconcentrated to yield a yellow oil, which was purified using flashchromatography using the solvent system described above to yield 3.6 g(50% yield) of a clear wax containing a mixture of compound 23 andmonoacylated products in a ratio of 5 to 1, as estimated from comparingthe integrals for the peaks for the (CH3)₃N—, —CH₂StBu and —CH₂COO—moieties. Analysis of the oil by HRMS (ESI-TOF) produced a prominent ionat m/z 820.4972, calculated for M+Na⁺═C₄₀H₈₀NNaO₈PS₂ ⁺820.4960.

A. Synthesis Variant 1 Phospholipase-A2 Treatment

Compound 24.

A mixture of compound 23 and monoacetylated products as described above(3.1 g, 3.9 mmol) was dissolved in Et₂O (400 mL) and methanol (30 mL).Borate buffer (100 mL, pH 7.4 0.1M, 0.072 mM in CaCl₂) was added,followed by phospholipase-A2 (from bee venom, 130 units, Sigma). Theresulting mixture was left to stir for 10 hr., at which point TLC(silica, MeOH:water 4:1—the previous solvent system 10:5:1DCM:MeOH:concentrated NH₄OH proved ineffective) showed the absence ofthe starting material (R_(f)=0.7) and the appearance of a new spot(R_(f)=0.2). The organic and aqueous layers were separated and theaqueous layer was washed with ether (2×250 mL). The product wasextracted from the aqueous layer with a mixture of DCM:MeOH (2:1, 2×50mL). The organic layers were then concentrated by rotary evaporation toyield product as a white wax (1.9 g, 86% yield) that NMR showed to be apure product (compound 24).

¹H NMR (CDCl₃, 500 MHz) δ 1.25-1.27 (br s, 12H), 1.31 (s, 9H), 1.35-1.45(m, 2H), 1.52-1.60 (m, 4H), 2.31 (t, 2H, J=7.5 Hz), 2.51 (t, 2H, J=7.5Hz), 3.28 (br s, 9H) 3.25-3.33 (br s, 2H), 3.78-3.86 (m, 1H), 3.88-3.96(m, 2H), 4.04-4.10 (m, 2H), 4.26-4.34 (m, 2H). Analysis of the wax byHRMS (ESI-TOF) produced a prominent ion at m/z 550.2936, calculated forM+Na⁺ 550.2943 (C₂₄H₅₀NNaO₇PS₂ ⁺), and an m/z at 528.3115, calculatedfor MH⁺ 528.3124 (C₂₄H₅₁NO₇PS₂ ⁺).

Anal. Calculated. for C₂₄H₅₀NO₇PS+2H₂O (563.73): C, 51.13; H, 9.66; N,2.48. Found: C, 50.90; H, 9.37; N, 2.76.

Compound 26.

The lyso compound 24 (1.5 g, 2.7 mmol) was dissolved in a mixture ofsec-butanol (5 mL) and Et₂O (200 mL), and the resulting cloudy mixturewas sonicated until the cloudiness dissipated. Buffer (200 mL, pH 5.8,0.2 M NaOAc, 0.08 M CaCl₂) was added, followed by cabbage extract (80 mLof extract from savoy cabbage (which contains phospholipase-D),containing 9 mg of protein/mL). The reaction was stirred for 1 day andmonitored by TLC (C₁₈RP SiO₂, 5:1 ACN:water), R_(f) of starting materialand product=0.3 and 0.05, respectively. In order to push the reaction tocompletion, as needed an additional portion of cabbage extract (50 mL)was added and the reaction stirred a further day. This process wasrepeated twice more, as needed to complete the conversion. When thereaction was complete, the mixture was concentrated on the rotaryevaporator to remove the ether, and then EDTA solution (0.5 M, 25 mL)was added and the product extracted into a 5:4 mixture of MeOH:DCM (300mL). Concentration of the organic layer followed by recrystallization ofthe residue from DCM and acetone afforded pure product (0.9 g, 75%yield). ¹H NMR (CDCl₃, 200 MHz) δ 1.25-1.27 (br s, 12H), 1.33 (s, 9H),1.52-1.60 (m, 4H), 2.34 (t, 2H, J=7.5 Hz), 2.52 (t, 2H, J=7.5 Hz),3.6-3.8 (br s, 1H), 3.85-3.97 (br s, 2H), 4.02-4.18 (m, 2H).

Compound 27.

The protected sample LPA, compound 26 (, 0.150 g, 0.34 mmol), wasmethanol washed and added to a vial in the glove box. This was thensuspended in a mixture of AcOH:THF (1:1, 10 mL), which never fullydissolved even after 1 hr. of sonication. Solid [Me₂SSMe]OTf (0.114 g,0.44 mmol) was then added. This was left to stir for 18 hr. The reactionwas monitored by removing an aliquot, concentrating it to dryness undervacuum, and re-dissolving or suspending the residue in CD₃OD forobserving the ¹H NMR shift of the CH₂ peak closest to the sulfur. Thestarting material had a peak at 2.52 ppm, whereas the unsymmetricaldisulfide formed at this juncture had a peak at around 2.7 ppm. Thismaterial (compound 27) was not further isolated or characterized.

Compound 28.

The mixture containing compound 27 was treated with water (100 μL)immediately followed by PMe₃ (0.11 g, 1.4 mmol). After stirring for 3hr. the solvent was removed by vacuum to yield an insoluble white solid.Methanol (5 mL) was added, the mixture centrifuged, and the motherliquor decanted. Vacuum concentration yielded 120 mg (91% yield) ofcompound 28, a beige solid. Compound 28 is a thiolated LPA hapten thatcan be conjugated to a carrier, for example, albumin or KLH, viadisulfide bond formation. Characterization of compound 28: ¹H NMR (1:1CD₃OD:CD₃CO₂D, 500 MHz) δ 1.25-1.35 (br s, 12H), 1.32-1.4 (m, 2H),1.55-1.6 (m, 4H), 2.34 (t, 2H, J=7), 2.47 (t, 2H, J=8.5), 3.89-3.97 (brs, 2H), 3.98-4.15 (m, 2H), 4.21 (m, 1H). Negative ion ES of the sampledissolved in methanol produced a predominant ion at m/z=385.1.

Example 4 Antibodies to S1P

One type of therapeutic antibody specifically binds undesirablesphingolipids to achieve beneficial effects such as, e.g., (1) loweringthe effective concentration of undesirable, toxic sphingolipids (and/orthe concentration of their metabolic precursors) that would promote anundesirable effect such as a cardiotoxic, tumorigenic, or angiogeniceffect; (2) to inhibit the binding of an undesirable, toxic,tumorigenic, or angiogenic sphingolipids to a cellular receptortherefore, and/or to lower the concentration of a sphingolipid that isavailable for binding to such a receptor. Examples of such therapeuticeffects include, but are not limited to, the use of anti-S1P antibodiesto lower the in vivo serum concentration of available S1P, therebyblocking or at least limiting S1P's tumorigenic and angiogenic effectsand its role in post-MI heart failure, cancer, or fibrongenic diseases.

Thiolated S1P (compound 10 of FIG. 1) was synthesized to contain areactive group (i.e., a sulfhydryl group) capable of cross-linking theessential structural features of S1P to a carrier moiety such as KLH.Prior to immunization, the thio-S1P analog was conjugated via IOA orSMCC cross-linking to protein carriers (e.g., KLH) using standardprotocols. SMCC is a heterobifunctional crosslinker that reacts withprimary amines and sulfhydryl groups, and represents a preferredcrosslinker.

Swiss Webster or BALB-C mice were immunized four times over a two monthperiod with 50 μg of immunogen (SMCC facilitated conjugate ofthiolated-S1P and KLH) per injection. Serum samples were collected twoweeks after the second, third, and fourth immunizations and screened bydirect ELISA for the presence of anti-S1P antibodies. Spleens fromanimals that displayed high titers of the antibody were subsequentlyused to generate hybridomas per standard fusion procedures. Theresulting hybridomas were grown to confluency, after which the cellsupernatant was collected for ELISA analysis. Of the 55 mice that wereimmunized, 8 were good responders, showing significant serum titers ofantibodies reactive to S1P. Fusions were subsequently carried out usingthe spleens of these mice and myeloma cells according to establishedprocedures. The resulting 1,500 hybridomas were then screened by directELISA, yielding 287 positive hybridomas. Of these 287 hybridomasscreened by direct ELISA, 159 showed significant titers. Each of the 159hybridomas was then expanded into 24-well plates. The cell-conditionedmedia of the expanded hybridomas were then re-screened to identifystable hybridomas capable of secreting antibodies of interest.Competitive ELISAs were performed on the 60 highest titer stablehybridomas.

Of the 55 mice and almost 1,500 hybridomas screened, one hybridoma wasdiscovered that displayed performance characteristics that justifiedlimited dilution cloning, as is required to ultimately generate a truemonoclonal antibody. This process yielded 47 clones, the majority ofwhich were deemed positive for producing S1P antibodies. Of these 47clones, 6 were expanded into 24-well plates and subsequently screened bycompetitive ELISA. From the 4 clones that remained positive, one waschosen to initiate large-scale production of the S1P monoclonalantibody. SCID mice were injected with these cells and the resultingascites was protein A-purified (50% yield) and analyzed for endotoxinlevels (<3 EU/mg). For one round of ascites production, 50 mice wereinjected, producing a total of 125 mL of ascites. The antibodies wereisotyped as IgG1 kappa, and were deemed >95% pure by HPLC. The antibodywas prepared in 20 mM sodium phosphate with 150 mM sodium chloride (pH7.2) and stored at −70° C.

The positive hybridoma clone (designated as clone 306D326.26) wasdeposited with the ATCC (safety deposit storage number SD-5362), andrepresents the first murine mAb directed against S1P. The clone alsocontains the variable regions of the antibody heavy and light chainsthat could be used for the generation of a “humanized” antibody variant,as well as the sequence information needed to construct a chimericantibody.

Screening of serum and cell supernatant for S1P-specific antibodies wasby direct ELISA using the thiolated S1P analog described in Example 1(i.e., compound 10) as the antigen. A standard ELISA was performed, asdescribed below, except that 50 ul of sample (serum or cell supernatant)was diluted with an equal volume of PBS/0.1% Tween-20 (PBST) during theprimary incubation. ELISAs were performed in 96-well high binding ELISAplates (Costar) coated with 0.1 μg of chemically-synthesized compound 10conjugated to BSA in binding buffer (33.6 mM Na₂CO₃, 100 mM NaHCO₃; pH9.5). The thiolated-S1P-BSA was incubated at 37° C. for 1 hr. at 4° C.overnight in the ELISA plate wells. The plates were then washed fourtimes with PBS (137 mM NaCl, 2.68 mM KCl, 10.14 mM Na₂HPO₄, 1.76 mMKH₂PO₄; pH 7.4) and blocked with PBST for 1 hr. at room temperature. Forthe primary incubation step, 75 uL of the sample (containing the S1P tobe measured), was incubated with 25 uL of 0.1 ug/mL anti-S1P mAb dilutedin PBST and added to a well of the ELISA plate. Each sample wasperformed in triplicate wells. Following a 1 hr. incubation at roomtemperature, the ELISA plates were washed four times with PBS andincubated with 100 ul per well of 0.1 ug/mL HRP goat anti-mousesecondary (Jackson Immunoresearch) for 1 hr. at room temperature. Plateswere then washed four times with PBS and exposed to tetramethylbenzidine(Sigma) for 1-10 minutes. The detection reaction was stopped by theaddition of an equal volume of 1M H₂SO₄. Optical density of the sampleswas determined by measurement at 450 nm using an EL-X-800 ELISA platereader (Bio-Tech).

For cross reactivity, a competitive ELISA was performed as describedabove, except for the following alterations (FIG. 4). The primaryincubation consisted of the competitor (S1P, SPH, LPA, etc.) and abiotin-conjugated anti-S1P mAb. Biotinylation of the purified monoclonalantibody was performed using the EZ-Link Sulfo-NHS-Biotinylation kit(Pierce). Biotin incorporation was determined as per kit protocol andranged from 7 to 11 biotin molecules per antibody. The competitor wasprepared as follows: lipid stocks were sonicated and dried under argonbefore reconstitution in DPBS/BSA [1 mg/ml fatty acid-free BSA(Calbiochem) in DPBS (Invitrogen 14040-133)]. Purified anti-S1P mAb wasdiluted as necessary in PBS/0.5% Triton X-100. Competitor and antibodysolutions were mixed together so to generate 3 parts competitor to 1part antibody. A HRP-conjugated streptavidin secondary antibody (JacksonImmunoresearch) was used to generate signal.

Another aspect of the competitive ELISA data shown in FIG. 4 is that itshows that the anti-S1P mAb was unable to distinguish the thiolated-S1Panalog (compound 10) from the natural S1P that was added in thecompetition experiment. It also demonstrates that the antibody does notrecognize any oxidation products because the analog was constructedwithout any double bonds (as is also true for the LPA analog describedin Example 3). The anti-S1P mAb was also tested against natural productcontaining the double bond that was allowed to sit at room temperaturefor 48 hours. Reverse phase HPLC of the natural S1P was performedaccording to methods reported previously (Deutschman, et al. (July2003), Am Heart J., vol. 146(1):62-8), and the results showed nodifference in retention time. Further, a comparison of the bindingcharacteristics of the monoclonal antibody to the various lipids shownin FIG. 4 indicates that the epitope recognized by the antibody do notinvolve the hydrocarbon chain in the region of the double bond ofnatural S1P. On the other hand, the epitope recognized by the monoclonalantibody is the region containing the amino alcohol on the sphingosinebase backbone plus the free phosphate. If the free phosphate is linkedwith a choline (as is the case with SPC), then the binding was somewhatreduced. If the amino group is esterified to a fatty acid (as is thecase with C1P), no antibody binding was observed. If the sphingosineamino alcohol backbone was replaced by a glycerol backbone (as is thecase with LPA), there the SIP-specific monoclonal exhibited no binding.These epitope mapping data indicate that there is only one epitope onS1P recognized by the monoclonal antibody, and that this epitope isdefined by the unique polar headgroup of S1P.

In a similar experiment using ELISA measurements, suitable controlmaterials were evaluated to ensure that this anti-S1P monoclonalantibody did not recognize either the protein carrier or thecrosslinking agent. For example, the normal crosslinker SMCC wasexchanged for IOA in conjugating the thiolated-S1P to BSA as the laydownmaterial in the ELISA. When IOA was used, the antibody's bindingcharacteristics were nearly identical to when BSA-SMCC-thiolated-S1P wasused. Similarly, KLH was exchanged for BSA as the protein that wascomplexed with thiolated-S1P as the laydown material. In thisexperiment, there was also no significant difference in the bindingcharacteristics of the antibody.

Binding Kinetics:

The binding kinetics of S1P to its receptor or other moieties has,traditionally, been problematic because of the nature of lipids. Manyproblems have been associated with the insolubility of lipids. ForBIAcore measurements, these problems were overcome by directlyimmobilizing S1P to a BIAcore chip. Antibody was then flowed over thesurface of the chip and alterations in optical density were measured todetermine the binding characteristics of the antibody to S1P. Tocircumvent the bivalent binding nature of antibodies, S1P was coated onthe chip at low densities. Additionally, the chip was coated withvarious densities of S1P (7, 20, and 1000 RU) and antibody binding datawas globally fit to a 1:1 interaction model. FIG. 5 demonstrates thechanges in optical density due to the binding of the monoclonal antibodyto S1P at three different densities of S1P. Overall, the affinity of themonoclonal antibody to S1P was determined to be very high, in the rangeof approximately 88 picomolar (pM) to 99 nM, depending on whether amonovalent or bivalent binding model was used to analyze the bindingdata.

Example 5 Cloning and Characterization of the Variable Domains of an S1PMonoclonal Antibody

A. Introduction.

The manufacture of biological products is complex, in part because ofthe complexity associated with the variability of the protein itself.For monoclonal antibodies (mAbs), variability can be localized to theprotein backbone or to the carbohydrate moieties appended to theseglycosylated proteins. For example, heterogeneity can be attributed tothe formation of alternative disulfide pairings, deamidation and theformation of isoaspartyl residues, methionine and cysteine oxidation,cyclization of N-terminal glutamine residues to pyroglutamate andpartial enzymatic cleavage of C-terminal lysines by mammaliancarboxypeptidases. On the other hand, carbohydrate heterogeneityintroduced during cell culture includes differential addition of fucose,alternative mannose branching linkages, and differential presence ofterminal sialylation. In addition, mutagenesis can be performed to alterglycosylation patterns. Oxidation is also a source of concern. Forinstance, the recombinant humanized monoclonal antibody HER2 undergoesoxidation in liquid formulations when exposed to intense light andelevated temperatures. Interestingly, such oxidation was reported to beformulation dependent (Lam, et al. (1997), Pharm. Sci., vol. 86:1250-1255), and the presence of NaCl in the formulation reportedlycaused an increase in oxidation at higher temperatures after contactwith stainless steel containers or stainless steel components in thefilling process. The methionine residue at position 255 in the heavychain of the Fc region was determined to be the primary site ofoxidation. The oxidation was eliminated by supplementing the media withmethionine and thiosulfate caused by free radicals generated by thepresence of metal ions and peroxide impurities in the formulation. Forreasons such as these, process engineering is commonly applied toantibody molecules to improve their properties, such as enhancedexpression in heterologous systems, resistance to proteases, reducedaggregation, and enhanced stability.

This example reports the cloning of the murine mAb against S1P. Thisantibody, termed Sphingomab™, is an IgG1 monoclonal antibody. Theoverall strategy consisted of cloning the murine variable domains ofboth the light chain (VL) and the heavy chain (VH). The consensussequence of 306D VH shows that the constant region fragment isconsistent with a gamma 2b isotype. The murine variable domains werecloned together with the constant domain of the light chain (CL) andwith the constant domain of the heavy chain (CH1, CH2, and CH3),resulting in a chimeric antibody construct. Also, Sphingomab™ is uniquebecause of the presence of a free cysteine residue in the Fab region atposition 50 on the heavy chain in the CDR2 region. Replacing thisresidue could greatly facilitate formulation and manufacturingprocesses, as well as improving yields. Indeed, in an effort to improvethe biophysical properties of the antibody molecule, substitution of thecysteine residue at position 50 with a panel of amino acid residues wasperformed by creating a series of constructs containing the desiredsubstitution. These constructs were then expressed in mammalian cells,and the different antibody variants were compared in an ELISA assay forbinding to S1P. Compared with the chimeric antibody, the resultingmutants carrying the substitution Cys50Ser and Cys50Arg exhibited aslight decrease in binding to S1P whereas the replacement of Cys withPhe or Ala did not alter the binding to S1P.

B. Materials and Methods.

1. Antibody Gene Cloning

A clone from the anti-S1P hybridoma cell line 306D326.1 (ATCC #SD-5362)was grown in DMEM (Dulbecco's Dulbecco's Modified Eagle Medium withGlutaMAX™ I, 4500 mg/L D-Glucose, Sodium Puruvate; Gibco/Invitrogen,Carlsbad, Calif., 111-035-003), 10% FBS (Sterile Fetal Clone I, PerbioScience), and 1× glutamine/Penicillin/Streptomycin (Gibco/Invitrogen).Total RNA was isolated from 10⁷ hybridoma cells using a procedure basedon the RNeasy Mini kit (Qiagen, Hilden Germany). The RNA was used togenerate first strand cDNA following the manufacturer's protocol (1^(st)strand synthesis kit, Amersham Biosciences).

The immunoglobulin heavy chain variable region (VH) cDNA was amplifiedby PCR using an MHV7 primer (MHV7: 5′-ATGGRATGGAGCKGGRTCTTTMTCTT-3′ [SEQID NO: 1]) in combination with a IgG2b constant region primerMHCG1/2a/2b/3 mixture (MHCG1: 5′-CAGTGGATAGACAGATGGGGG-3′ [SEQ ID NO:2]; MHCG2a: 5′-CAGTGGATAGACCGATGGGGC-3 [SEQ ID NO: 3]; MHCG2b:5′-CAGTGGATAGACTGATGGGGG-3′ [SEQ ID NO: 4]; MHCG3:5′-CAAGGGATAGACAGATGGGGC-3′ [SEQ ID NO: 5]). The product of the reactionwas ligated into the pCR2.1®-TOPO® vector (Invitrogen) using the TOPO-TACloning® kit and sequence. The variable domain of the heavy chain wasthen amplified by PCR from this vector and inserted as a Hind III andApa I fragment and ligated into the expression vector pG1D200 (see U.S.Pat. No. 7,060,808) or pG4D200 (id.) containing the HCMVi promoter, aleader sequence, and the gamma-1 constant region to generate the plasmidpG1D200306DVH. The consensus sequence of 306D V_(H) (FIG. 6; SEQ ID NO:6) showed that the constant region fragment was consistent with a gamma2b isotype.

Similarly, the immunoglobulin kappa chain variable region (VK) wasamplified using the MKV 20 primer (5′-GTCTCTGATTCTAGGGCA-3′ [SEQ ID NO:7]) in combination with the kappa constant region primer MKC(5′-ACTGGATGGTGGGAAGATGG-3′ [SEQ ID NO: 8]). The product of thisreaction was ligated into the pCR2.1®-TOPO® vector using the TOPO-TACloning® kit and sequence. The variable domain of the light chain wasthen amplified by PCR and then inserted as a Bam HI and Hind IIIfragment into the expression vector pKN100 (see U.S. Pat. No. 7,060,808)containing the HCMV promoter, a leader sequence, and the human kappaconstant domain, generating plasmid pKN100306DVK.

The heavy and light chain plasmids pG1D200306DVH plus pKN100306DVK weretransformed into DH4a bacteria and stocked in glycerol. Large-scaleplasmid DNA was prepared as described by the manufacturer (Qiagen,endotoxin-free MAXIPREP™ kit). DNA samples, purified using Qiagen'sQIAprep Spin Miniprep Kit or EndoFree Plasmid Mega/Maxi Kit, weresequenced using an ABI 3730xl automated sequencer, which also translatesthe fluorescent signals into their corresponding nucleobase sequence.Primers were designed at the 5′ and 3′ ends so that the sequenceobtained would overlap. The length of the primers was 18-24 bases, andpreferably they contained 50% GC content and no predicted dimers orsecondary structure. The amino acid sequences for the mouse V_(H) andV_(L) domains from Sphingomab™ are shown in FIG. 6 (SEQ ID NOS: 6 and 9,respectively). In FIG. 6, the CDR residues (see Kabat, E A (1982),Pharmavol Rev, vol. 34: 23-38) are boxed, and are shown below in Table1.

TABLE 1 Mouse Sphingomab ™ CDR sequences of the mouse V_(H) and V_(L)domains CDR VL CDR ITTTDIDDDMN (SEQ ID NO: 10) CDR1 EGNILRP (SEQ ID NO:11) CDR2 LQSDNLPFT (SEQ ID NO: 12) CDR3 VH CDR DHTIH (SEQ ID NO: 13)CDR1 CISPRHDITKYNEMFRG (SEQ ID NO: 14) CDR2 GGFYGSTIWFDF (SEQ ID NO: 15)CDR3

The complete nucleotide and amino acid sequences of several chimericantibody V_(H) and V_(L) domains are shown in FIG. 7. In FIG. 7, theamino acid sequences are numbered, and the CDRs identified, according tothe Kabat method (Kabat, et al. (1991), NIH National TechnicalInformation Service, pp. 1-3242).

2. COS 7 Expression

For antibody expression in a non-human mammalian system, plasmids weretransfected into the African green monkey kidney fibroblast cell lineCOS 7 by electroporation (0.7 ml at 10⁷ cells/ml) using 10 ug of eachplasmid. Transfected cells were plated in 8 ml of growth medium for 4days. The chimeric 306DH1×306DVK-2 antibody was expressed at 1.5 μg/mlin transiently co-transfected COS cell conditioned medium. The bindingof this antibody to S1P was measured using the S1P ELISA.

The expression level of the chimeric antibody was determined in aquantitative ELISA as follows. Microtiter plates (Nunc MaxiSorpimmunoplate, Invitrogen) were coated with 100 μl aliquots of 0.4 μg/mlgoat anti-human IgG antibody (Sigma, St. Louis, Mo.) diluted in PBS andincubate overnight at 4° C. The plates were then washed three times with200 μl/well of washing buffer (1×PBS, 0.1% TWEEN). Aliquots of 200 μL ofeach diluted serum sample or fusion supernatant were transferred to thetoxin-coated plates and incubated for 37° C. for 1 hr. Following 6washes with washing buffer, the goat anti-human kappa light chainperoxidase conjugate (Jackson Immuno Research) was added to each well ata 1:5000 dilution. The reaction was carried out for 1 hr at roomtemperature, plates were washed 6 times with the washing buffer, and 150μL of the K-BLUE substrate (Sigma) was added to each well, incubated inthe dark at room temperature for 10 min. The reaction was stopped byadding 50 μl of RED STOP solution (SkyBio Ltd.) and the absorption wasdetermined at 655 nm using a Microplater Reader 3550 (Bio-RadLaboratories Ltd.). Results from the antibody binding assays are shownin FIG. 8.

3. 293F Expression

For antibody expression in a human system, plasmids were transfectedinto the human embryonic kidney cell line 293F (Invitrogen) using293fectin (Invitrogen) and using 293F-FreeStyle Media (Invitrogen) forculture. Light and heavy chain plasmids were both transfected at 0.5g/mL. Transfections were performed at a cell density of 10⁶ cells/mL.Supernatants were collected by centrifugation at 1100 rpm for 5 minutesat 25° C. 3 days after transfection. Expression levels were quantifiedby quantitative ELISA (see below) and varied from 0.25-0.5 g/mL for thechimeric antibody.

4. Quantitative ELISA

Microtiter ELISA plates (Costar) were coated with rabbit anti-mouse IgG,F(ab′)₂ fragment specific (Jackson Immuno Research) or rabbitanti-human, IgG F(ab′)₂ fragment specific (Jackson Immuno Research)diluted in 1 M Carbonate Buffer (pH 9.5) at 37° C. for 1 hr. Plates werewashed with PBS and blocked with PBS/BSA/Tween-20 for 1 hr at 37° C. Forthe primary incubation, dilutions of non-specific mouse IgG or humanIgG, whole molecule (used for calibration curve) and samples to bemeasured were added to the wells. Plates were washed and incubated with100 ul per well of HRP conjugated goat anti-mouse (H+L) diluted 1:40,000(Jackson Immuno Research) or HRP conjugated goat anti-human (H+L)diluted 1:50,000 (Jackson Immuno Research) for 1 hr at 37° C. Afterwashing, the enzymatic reaction was detected with Tetramethylbenzidine(Sigma) and stopped by adding 1 M H₂SO₄. The optical density (OD) wasmeasured at 450 nm using a Thermo Multiskan EX. Raw data weretransferred to GraphPad software for analysis.

5. Direct ELISA

Microtiter ELISA plates (Costar) were coated overnight with S1P dilutedin 1 M Carbonate Buffer (pH 9.5) at 37° C. for 1 hr. Plates were washedwith PBS (137 mM NaCl, 2.68 mM KCl, 10.1 mM Na₂HPO₄, 1.76 mM KH₂PO₄; pH7.4) and blocked with PBS/BSA/Tween-20 for 1 hr at room temp orovernight at 4° C. For the primary incubation (1 hr at room temp.), astandard curve using the anti-S1P mAb and the samples to be tested forbinding was built using the following set of dilutions: 0.4 μg/mL, 0.2μg/mL, 0.1 μg/mL, 0.05 μg/mL, 0.0125 μg/mL, and 0 μg/mL, and 100 μladded to each well. Plates were washed and incubated with 100 μl perwell of HRP conjugated goat anti-mouse (1:20,000 dilution) (JacksonImmuno Research) or HRP conjugated goat anti-human (H+L) diluted1:50,000 (Jackson Immuno Research) for 1 hr at room temperature. Afterwashing, the enzymatic reaction was detected with tetramethylbenzidine(Sigma) and stopped by adding 1 M H₂SO₄. The optical density (OD) wasmeasured at 450 nm using a Thermo Multiskan EX. Raw data weretransferred to GraphPad software for analysis.

Table 2, below, shows a comparative analysis of mutants with thechimeric antibody. To generate these results, bound antibody wasdetected by a second antibody, specific for the mouse or human IgG,conjugated with HRP. The chromogenic reaction was measured and reportedas Optical density (OD). The concentration of the panel of antibodieswas 0.1 ug/ml. No interaction of the second antibody with S1P-coatedmatrix alone was detected.

TABLE 2 Variable Domain Mutation Plasmids Binding Chimeric pATH50 + pATH10 1.5 HC CysAla pATH50 + pATH11C1 2 CysSer pATH50 + pATH 0.6 12C2CysArg pATH50 + pATH14C1 0.4 CysPhe pATH50 + pATH16C1 2 LC MetLeupATH53C1 + pATH10 1.6

Example 6 Chimeric mAb to S1P

As used herein, the term “chimeric” antibody (or “immunoglobulin”)refers to a molecule comprising a heavy and/or light chain which isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (Cabilly et al., supra; Morrison et al.,Proc. Natl. Acad. Sci. U.S.A. 81:6851 (1984)).

A chimeric antibody to S1P was generated using the variable regions (Fv)containing the active S1P binding regions of the murine antibody from aparticular hybridoma (ATCC safety deposit storage number SD-5362) withthe Fc region of a human IgG1 immunoglobulin. The Fc regions containedthe CL, ChL, and Ch3 domains of the human antibody. Without beinglimited to a particular method, chimeric antibodies could also have beengenerated from Fc regions of human IgG1, IgG2, IgG3, IgG4, IgA, or IgM.As those in the art will appreciate, “humanized” antibodies can beengenerated by grafting the complementarity determining regions (CDRs,e.g. CDR1-4) of the murine anti-S1P mAb with a human antibody frameworkregions (e.g., Fr1, Fr4, etc.) such as the framework regions of an IgG1.FIG. 9 shows the binding of the chimeric and full murine mAbs in adirect ELISA measurement using thiolated-S1P as lay down material.

For the direct ELISA experiments shown in FIG. 9, the chimeric antibodyto S1P had similar binding characteristics to the fully murinemonoclonal antibody. ELISAs were performed in 96-well high-binding ELISAplates (Costar) coated with 0.1 ug of chemically-synthesized, thiolatedS1P conjugated to BSA in binding buffer (33.6 mM Na₂CO₃, 100 mM NaHCO₃;pH 9.5). The thiolated S1P-BSA was incubated at 37° C. for 1 hr. or at4° C. overnight in the ELISA plate. Plates were then washed four timeswith PBS (137 mM NaCl, 2.68 mM KCl, 10.14 mM Na₂HPO₄, 1.76 mM KH₂PO₄; pH7.4) and blocked with PBST for 1 hr. at room temperature. For theprimary incubation step, 75 uL of the sample (containing the S1P to bemeasured), was incubated with 25 μL of 0.1 μg/mL anti-S1P monoclonalantibody diluted in PBST and added to a well of the ELISA plate. Eachsample was performed in triplicate wells. Following a 1 hr incubation atroom temperature, the ELISA plates were washed four times with PBS andincubated with 100 ul per well of 0.1 ug/mL HRP goat anti-mousesecondary (Jackson Immunoresearch) for 1 hr. at room temperature. Plateswere then washed four times with PBS and exposed to tetramethylbenzidine(Sigma) for 1-10 minutes. The detection reaction was stopped by theaddition of an equal volume of 1M H₂SO₄. Optical density of the sampleswas determined by measurement at 450 nm using an EL-X-800 ELISA platereader (Bio-Tech).

As was the case with regard to the experiments described in Example 4,the preferred method of measuring either antibody titer in the serum ofan immunized animal or in cell-conditioned media (i.e., supernatant) ofan antibody-producing cell such as a hybridoma, involves coating theELISA plate with a target ligand (e.g., a thiolated analog of S1P, LPA,etc.) that has been covalently linked to a protein carrier such as BSA.

Without being limited to particular method or example, chimericantibodies could be generated against other lipid targets such as LPA,ceramides, sulfatides, cerebrosides, cardiolipins, phosphotidylserines,phosphotidylinositols, phosphatidic acids, phosphotidylcholines,phosphatidylethanolamines, eicosinoids, and other leukotrienes, etc.Further, many of these lipids could also be glycosylated and/oracetylated, if desired.

Example 7 Antibody-Based Assay for Sphingosine Kinase (SPH Kinase)

Sphingosine Kinase (SPH kinase or SPHK) catalyzes the conversion of SPHto S1P. A genetic sequence encoding human SPH-kinase has been described(Melendez et al., Gene 251:19-26, 2000). Three human homologs of SPHkinase (SKA, SKB, and SKC) have been described (published PCT patentapplication WO 00/52173). Murine SPH kinase has also been described(Kohama et al., J. Biol. Chem. 273:23722-23728, 1998; and published (PCTpatent application WO 99/61581). Published PCT patent application WO99/61581 reports nucleic acids encoding a sphingosine kinase. PublishedPCT patent application WO 00/52173 reports nucleic acids encodinghomologues of sphingosine kinase. Other SPH kinases have also beenreported. See, e.g., Pitson et al., Biochem J. 350:429-441, 2000;published PCT application WO 00/70028; Liu et al., J. Biol. Chem.,275:19513-19520, 2000; PCT/AU01/00539, published as WO 01/85953;PCT/US01/04789, published as WO 01/60990; and PCT/EP00/09498, publishedas WO 01/31029.

Inhibitors of SPH kinase include, but are not limited to,N,N-dimethylsphingosine (Edsall et al., Biochem. 37:12892-12898, 1998);D-threo-dihydrosphingosine (Olivera et al., Nature 365:557-560, 1993);and Sphingoid bases (Jonghe et al, “Structure-Activity Relationship ofShort-Chain Sphingoid Bases As Inhibitors of Sphingosine Kinase”,Bioorganic & Medicinal Chemistry Letters 9:3175-3180, 1999)

Assays of SPH kinase useful for evaluating these and other known orpotential SPH kinase inhibitors include those disclosed by Olivera etal., Methods in Enzymology, 311:215-223, 1999; Caligan et al.,Analytical Biochemistry, 281:36-44, 2000.

Inhibition of SPH kinase is believed to lead to an accumulation of itssubstrate, SPH, which, like S1P, can be an undesirable sphingolipid incertain conditions. In order to avoid or mitigate these undesirableeffects, an agent could be administered that (i) stimulates an enzymethat utilizes SPH as a substrate, provided that the enzyme should not beone that yields S1P as a reaction product (such as, e.g., ceramidesynthase; see below); or (ii) inhibits an enzyme that yields SPH as aproduct.

Without being limited to a particular method, anti-S1P antibodies (e.g.,a monoclonal anti-S1P antibody) could be used as a reagent in an invitro assay for SPH kinase activity. For example, purified SPHK could beadded to the wells of a microtiter plate in the presence of PBS and thesubstrate for the kinase, SPH (complexed with, for example, fatty-acidfree BSA). The resulting product of the reaction, S1P, could then befollowed by ELISA using an anti-S1P antibody (e.g., the monoclonalanti-S1P antibody described above in Example 4). In such an assay,inhibition of SPHK by a test compound would result in lower levels ofS1P than in a control reaction that did not include an SPHK inhibitorycompound. Such an assay could be configured for high throughput, andcould thus serve as the basis of a high throughput screening assay formodulators of SPHK activity.

Example 8 Antibody-Based Assay for S1P Lyase or SPP Activities

The stimulation of enzymes that catalyze reactions that degrade S1P(i.e., reactions that utilize S1P as a reactant) will result in thestimulation of degradation of S1P molecules. Such enzymes include, butare not limited to:

S-1-P Lyase:

S1P lyase catalyzes the conversion of S1P to ethanolamine-P (also knownas t-2-hexadecanal) and palmitaldehyde (Veldhoven et al., Adv. LipidRes. 26:67-97, 1993; Van Veldhoven, Methods in Enzymology, 311:244-254,1999). Yeast (Lanterman et al, Biochem. J. 332:525-531, 1998), murine(Zhou et al., Biochem. Biophys. Res. Comm. 242:502-507, 1998), and human(published PCT patent application WO 99/38983) S1P lyase genes have beenreported. Published PCT patent application WO 99/16888 reports S1P lyaseDNA and protein sequences. U.S. Pat. No. 6,187,562 and published PCTpatent application WO 99/38983 also report an S1P lyase.

Gain-of-function assays can be developed to discover small moleculecompounds that would activate the lyase or increase the expression ofthe gene encoding it. Without being limited to a particular method, onecould use anti-S1P antibodies in an ELISA format to measure theproduction of S1P from added SPH in in vitro or cell-based formats.Compounds identified as stimulating S1P lyase activity, either directlyat the enzyme or indirectly by elevating the expression level of thegene encoding the enzyme (for example, by gene activation, enhancing S1Plyase mRNA stability, etc.), could be investigated further, as suchcompounds may prove useful in lowering the extracellular concentrationof S1P in patients where S1P levels correlate with toxicity, such as inthe treatment of cancer, cardio and cerebrovascular disease, autoimmunedisorders, inflammatory disorders, angiogenesis, fibrotic diseases, andage-related macular degeneration.

S1P Phosphatase:

S1P phosphatase (also known as SPP phosphohydrolase) is a mammalianenzyme that catalyzes the conversion of S-1-P to sphingosine (Mandala etal., Proc. Nat. Acad. Sci. 95:150-155, 1998; Mandala et al, Proc. Nat.Acad. Sci. 97:7859-7864, 2000; Mandala, Prostaglandins & other LipidMediators, 64:143-156, 2001; Brindley et al., Methods in Enzymology,311:233-244, 1999). Two S-1-P phosphatases, LBP1 and LBP2, have beenisolated from yeast (Mandala et al., J. Biol. Chem. 272:32709-32714,1997); PCT/UW01/03879, published as WO01/57057.

As with S1P lyase, gain-of-function assays can be developed to discovercompounds that would activate S1P phosphatase or increase the expressionof the gene encoding it. For example, one can use anti-S1P antibodies inan ELISA format to measure the production of S1P from added SPH in invitro or cell-based formats. Compounds identified as stimulating S1Pphosphatase activity, either directly at the enzyme or indirectly byelevating the expression level of the gene encoding the enzyme (forexample, by gene activation, enhancing S1P phosphatase mRNA stability,etc.), could be investigated further, as such compounds may prove usefulin lowering the extracellular concentration of S1P in patients where S1Plevels correlate with toxicity, such as cancer, cardio andcerebrovascular disease, autoimmune disorders, inflammatory disorders,angiogenesis, fibrotic diseases, and age-related macular degeneration.

Example 9 Production and Characterization of Monoclonal Antibodies toLPA

Antibody Production

Although polyclonal antibodies against naturally-occurring LPA have beenreported in the literature (Chen J H, et al., Bioorg Med Chem. Lett.2000 Aug. 7; 10(15):1691-3), monoclonal antibodies have not beendescribed. Using an approach similar to that described in Example 4, aC-12 thio-LPA analog (compound 28 in Example 3) as the key component ofa hapten formed by the cross-linking of the analog via the reactive SHgroup to a protein carrier (KLH) via standard chemical cross-linkingusing either IOA or SMCC as the cross-linking agent, monoclonalantibodies against LPA were generated. To do this, mice were immunizedwith the thio-LPA-KLH hapten (in this case, thiolated-LPA:SMCC:KLH)using methods described in Example 4 for the generation of anti-S1Pmonoclonal antibodies. Of the 80 mice immunized against the LPA analog,the five animals that showed the highest titers against LPA (determinedusing an ELISA in which the same LPA analog (compound 28) as used in thehapten was conjugated to BSA using SMCC and laid down on the ELISAplates) were chosen for moving to the hybridoma phase of development.

The spleens from these five mice were harvested and hybridomas weregenerated by standard techniques. Briefly, one mouse yielded hybridomacell lines (designated 504A). Of all the plated hybridomas of the 504Aseries, 66 showed positive antibody production as measured by thepreviously-described screening ELISA.

Table 3, below, shows the antibody titers in cell supernatants ofhybridomas created from the spleens of two of mice that responded to anLPA analog hapten in which the thiolated LPA analog was cross-linked toKLH using heterobifunctional cross-linking agents. These datademonstrate that the anti-LPA antibodies do not react either to thecrosslinker or to the protein carrier. Importantly, the data show thatthe hybridomas produce antibodies against LPA, and not against S1P.

TABLE 3 LPA hybridomas LPA 3rd bleed titer Supernatants binding S1Pbinding Cross reactivity mouse # OD at 1:312,500 from 24 well OD at 1:20OD at 1:20 w/ S1P* 1 1.242 1.A.63 1.197 0.231 low 1.A.65 1.545 0.176none 2 0.709 2.B.7 2.357 0.302 low 2.B.63 2.302 0.229 low 2.B.83 2.7120.175 none 2.B.104 2.57 0.164 none 2.B.IB7 2.387 0.163 none 2.B.3A62.227 0.134 none *Cross reactivity with S1P from 24 well supernatantshigh = OD > 1.0-2.0 at [1:20] mid = OD 0.4-1.0 at [1:20] low = OD0.4-0.2 at [1:20] none = OD < 0.2 OD at [1:20]

The development of anti-LPA mAbs in mice was monitored by ELISA (directbinding to 12:0 and 18:1 LPA and competition ELISA). A significantimmunological response was observed in at least half of the immunizedmice and five mice with the highest antibody titer were selected toinitiate hybridoma cell line development following spleen fusion.

After the initial screening of over 2000 hybridoma cell lines generatedfrom these 5 fusions, a total of 29 anti-LPA secreting hybridoma celllines exhibited high binding to 18:1 LPA. Of these hybridoma cell lines,24 were further subcloned and characterized in a panel of ELISA assays.From the 24 clones that remained positive, six hybridoma clones wereselected for further characterization. Their selection was based ontheir superior biochemical and biological properties.

Direct Binding Kinetics

The binding of 6 anti-LPA mAbs (B3, B7, B58, A63, B3A6, D22) to 12:0 and18:1 LPA (0.1 uM) was measured by ELISA. EC₅₀ values were calculatedfrom titration curves using 6 increasing concentrations of purified mAbs(0 to 0.4 ug/ml). EC₅₀ represents the effective antibody concentrationwith 50% of the maximum binding. Max denotes the maximal binding(expressed as OD450). Results are shown in Table 4.

TABLE 4 Direct Binding Kinetics of Anti-LPA mAbs B3 B7 B58 D22 A63 B3A612:0 LPA EC₅₀ (nM) 1.420 0.413 0.554 1.307 0.280 0.344 Max (OD450) 1.8091.395 1.352 0.449 1.269 1.316 18:1 LPA EC₅₀ (nM) 1.067 0.274 0.245 0.1760.298 0.469 Max (OD450) 1.264 0.973 0.847 0.353 1.302 1.027

The kinetics parameters k_(a) (association rate constant), k_(d)(disassociation rate constant) and K_(D) (association equilibriumconstant) were determined for the 6 lead candidates using the BIAcore3000 Biosensor machine. In this study, LPA was immobilized on the sensorsurface and the anti-LPA mAbs were flowed in solution across thesurface. As shown, all six mAbs bound LPA with similar K_(D) valuesranging from 0.34 to 3.8 μM and similar kinetic parameters.

The Anti-LPA Murine mAbs Exhibit High Affinity to LPA

LPA was immobilized to the sensor chip at densities ranging 150resonance units. Dilutions of each mAb were passed over the immobilizedLPA and kinetic constants were obtained by nonlinear regression ofassociation/dissociation phases. Errors are given as the standarddeviation using at least three determinations in duplicate runs.Apparent affinities were determined by K_(D)=k_(a)/k_(d).k_(a)=Association rate constant in M⁻¹s⁻¹ k_(d)=Dissociation rateconstant in s⁻¹

TABLE 5 Affinity of anti-LPA mAb for LPA mAbs k_(a) (M⁻¹ s⁻¹) k_(d)(s⁻¹) K_(D) (pM) A63 4.4 ± 1.0 × 10⁵ 1 × 10⁻⁶ 2.3 ± 0.5 B3 7.0 ± 1.5 ×10⁵ 1 × 10⁻⁶ 1.4 ± 0.3 B7 6.2 ± 0.1 × 10⁵ 1 × 10⁻⁶ 1.6 ± 0.1 D22 3.0 ±0.9 × 10⁴ 1 × 10⁻⁶ 33 ± 10 B3A6 1.2 ± 0.9 × 10⁶ 1.9 ± 0.4 × 10⁻⁵  16 ±1.2Specificity Profile of Six Anti-LPA mAbs.

Many isoforms of LPA have been identified to be biologically active andit is preferable that the mAb recognize all of them to some extent to beof therapeutic relevance. The specificity of the anti-LPA mAbs wasevaluated utilizing a competition assay in which the competitor lipidwas added to the antibody-immobilized lipid mixture. Competition ELISAassays were performed with 6 mAbs to assess their specificity. 18:1 LPAwas captured on ELISA plates. Each competitor lipid (up to 10 uM) wasserially diluted in BSA (1 mg/ml)-PBS and then incubated with the mAbs(3 nM). Mixtures were then transferred to LPA coated wells and theamount of bound antibody was measured with a secondary antibody. Dataare normalized to maximum signal (A₄₅₀) and are expressed as percentinhibition. Assays were performed in triplicate. IC₅₀: Half maximuminhibition concentration; MI: Maximum inhibition (% of binding in theabsence of inhibitor); ---: not estimated because of weak inhibition. Ahigh inhibition result indicates recognition of the competitor lipid bythe antibody. As shown in Table 6, all the anti-LPA mAbs recognized thedifferent LPA isoforms.

TABLE 6 Specificity profile of six anti-LPA mAbs. 14:0 LPA 16:0 LPA 18:1LPA 18:2 LPA 20:4 LPA IC₅₀ MI IC₅₀ MI IC₅₀ MI IC₅₀ MI IC₅₀ MI uM % uM %uM % uM % uM % 504B3 0.02 72.3 0.05 70.3 0.287 83 0.064 72.5 0.02 67.1504B7 0.105 61.3 0.483 62.9 >2.0 100 1.487 100 0.161 67 504B58-3F8 0.2663.9 5.698 >100 1.5 79.3 1.240 92.6 0.304 79.8 504B104 0.32 23.1 1.55726.5 28.648 >100 1.591 36 0.32 20.1 504D22-1 0.164 34.9 0.543 31 1.48947.7 0.331 31.4 0.164 29.5 504A63-1 1.147 31.9 5.994 45.7 — — — — 0.11914.5 504B3A6-1 0.108 59.9 1.151 81.1 1.897 87.6 — — 0.131 44.9Interestingly, the anti-LPA mAbs were able to discriminate between 12:0(lauroyl), 14:0 (myristoyl), 16:0 (palmitoyl), 18:1 (oleoyl), 18:2(linoleoyl) and 20:4 (arachidonoyl) LPAs. The rank order for EC₅₀ wasfor the unsaturated 18:2>18:1>20:4 and for the saturated lipids14:0>16:0>18:0. mAbs with high specificity are desirable for ultimatedrug development. The specificity of the anti-LPA mAbs was assessed fortheir binding to LPA related biolipids such as distearoyl-phosphatidicacid, lysophosphatidylcholine, S1P, ceramide and ceramide-1-phosphate.None of the six antibodies demonstrated crossreactivity to distearoyl PAand LPC, the immediate metabolic precursor of LPA.

Example 10 Anti-Cancer Activities of anti-LPA Monoclonal Antibodies

Cancer Cell Proliferation

LPA is a potent growth factor supporting cell survival and proliferationby stimulation of G_(i), G_(q) and G_(12/13) via GPCR-receptors andactivation of downstream signaling events. Cell lines were tested fortheir proliferative response to LPA (0.01 mM to 10 mM). Cellproliferation was assayed by using the cell proliferation assay kit fromChemicon (Temecula Calif.) (Panc-1) and the Cell-Blue titer from Pierce(Caki-1). Each data point is the mean of three independent experiments.LPA increased proliferation of 7 human-derived tumor cell lines in adose dependent manner including SKOV3 and OVCAR3 (ovarian cancer),Panc-1 (pancreatic cancer), Caki-1 (renal carcinoma cell), DU-145(prostate cancer), A549 (lung carcinoma), and HCT-116 (colorectaladenocarcinoma) cells and one rat-derived tumor cell line, RBL-2H3 (ratleukemia cells). Even though tumor-derived cells normally have highbasal levels of proliferation, LPA appears to further augmentproliferation in most tumor cell lines. Anti-LPA mAbs (B7 and B58) wereassessed for the ability to inhibit LPA-induced proliferation inselected human cancer cell lines. The increase in proliferation inducedby LPA was shown to be mitigated by the addition of anti-LPA mAb.

Anti-LPA mAb Sensitizes Tumor Cells to Chemotherapeutic Agents

The ability of LPA to protect ovarian tumor cells against apoptosis whenexposed to clinically-relevant levels of the chemotherapeutic agent,paclitaxel (Taxol) was investigated. SKVO3 cells were treated with 1%FBS (S), Taxol (0.5 mM), +/−anti-LPA mAbs for 24 h. LPA protected SKVO3cells from Taxol-induced apoptosis. Apoptosis was assayed by measurementof the caspase activity as recommended by the manufacturer (Promega). Asanticipated, LPA protected most of the cancer cell lines tested fromtaxol-induced cell death. When anti-LPA antibody was added to aselection of the LPA responsive cells, the anti-LPA antibody blocked theability of LPA to protect cells from death induced by the cytotoxicchemotherapeutic agent. Moreover, the anti-LPA antibody was able toremove the protection provided by serum. Serum is estimated to containabout 5-20 mM LPA. Taxol induced caspase-3,7 activation in SKOV3 cellsand the addition of serum to cells protected cells from apoptosis.Taxol-induced caspase activation was enhanced by the addition of all 3of the anti-LPA mAbs to the culture medium. This suggests that theprotective and anti-apoptotic effects of LPA were removed by theselective antibody mediated neutralization of the LPA present in serum.

Anti-LPA mAb Inhibits LPA-Mediated Migration of Tumor Cells

An important characteristic of metastatic cancers is that the tumorcells escape contact inhibition and migrate away from their tissue oforigin. LPA has been shown to promote metastatic potential in severalcancer cell types. Accordingly, we tested the ability of anti-LPA mAb toblock LPA-dependent cell migration in several human cancer cell lines byusing the cell monolayer scratch assay. Cells were seeded in 96 wellplates and grown to confluence. After 24 h of starvation, the center ofthe wells was scratched with a pipette tip. In this art-accepted“scratch assay,” the cells respond to the scratch wound in the cellmonolayer in a stereotyped fashion by migrating toward the scratch andclose the wound. Progression of migration and wound closure aremonitored by digital photography at 10× magnification at desiredtimepoints. Cells were not treated (NT), treated with LPA (2.5 mM) withor w/o mAb B7 (10 μg/ml) or an isotype matching non-specific antibody(NS) (10 μg/ml). In untreated cells, a large gap remains between themonolayer margins following the scratch. LPA-treated cells in contrast,have only a small gap remaining at the same timepoint, and a few cellsare making contact across the gap. In cells treated with both LPA andthe anti-LPA antibody B7, the gap at this timepoint was several foldlarger than the LPA-only treatment although not as large as theuntreated control cells. This shows that the anti-LPA antibody had aninhibitory effect on the LPA-stimulated migration of renal cellcarcinoma (Caki-1) cells. Similar data were obtained with mAbs B3 andB58. This indicates that the anti-LPA mAb can reduce LPA-mediatedmigration of cell lines originally derived from metastatic carcinoma.

Anti-LPA mAbs Inhibit Release of Pro-Tumorigenic Cytokines from TumorCells

LPA is involved in the establishment and progression of cancer byproviding a pro-growth tumor microenvironment and promotingangiogenesis. In particular, increases of the pro-growth factors such asIL-8 and VEGF have been observed in cancer cells. IL-8 is stronglyimplicated in cancer progression and prognosis. IL-8 may exert itseffect in cancer through promoting neovascularization and inducingchemotaxis of neutrophils and endothelial cells. In addition,overexpression of IL-8 has been correlated to the development of a drugresistant phenotype in many human cancer types.

Three anti-LPA mAbs (B3, B7 and B58) were tested for their abilities toreduce in vitro IL-8 production compared to a non-specific antibody(NS). Caki-1 cells were seeded in 96 well plates and grown toconfluency. After overnight serum starvation, cells were treated with18:1 LPA (0.2 mM) with or without anti-LPA mAb B3, B7, B58 or NS(Non-Specific). After 24 h, cultured supernatants of renal cancer cells(Caki-1), treated with or without LPA and in presence of increasingconcentrations of the anti-LPA mAbs B3, B7 and B58, were collected andanalyzed for IL-8 levels using a commercially available ELISA kit (HumanQuantikine Kit, R&D Systems, Minneapolis, Minn.). In cells pre-treatedwith the anti-LPA mAbs, IL-8 expression was significantly reduced in adose-dependent manner (from 0.1-30 μg/mL mAb) whereas LPA increased theexpression of IL-8 by an average of 100% in non-treated cells. Similarresults were obtained with the other well-known pro-angiogenic factor,VEGF. The inhibition of IL-8 release by the anti-LPA mAbs was alsoobserved in other cancerous cell lines such as the pancreatic cell linePanc-1. These data suggest that the blockade of the pro-angiogenicfactor release is an additional and potentially important effect ofthese anti-LPA mAbs.

Anti-LPA mAbs Inhibit Angiogenesis In Vivo

One of the anti-LPA mAbs (B7) was tested for its ability to mitigateangiogenesis in vivo using the Matrigel Plug assay. This assay utilizesMatrigel, a proprietary mixture of tumor remnants including basementmembranes derived from murine tumors. When Matrigel, or its derivategrowth factor-reduced (GFR) Matrigel, is injected sc into an animal, itsolidifies and forms a ‘plug.’ If pro-angiogenic factors are mixed withthe matrix prior to placement, the plug will be invaded by vascularendothelial cells which eventually form blood vessels. Matrigel can beprepared either alone or mixed with recombinant growth factors (bFGF,VEGF), or tumor cells and then injected sc in the flanks of 6-week oldnude (NCr Nu/Nu) female mice. In this example, Caki-1 (renal carcinoma)cells were introduced inside the Matrigel and are producing sufficientlevels of VEGF and/or IL8 and LPA. Matrigel plugs were preparedcontaining 5×10⁵ Caki-1 cells from mice treated with saline or with 10mg/kg of anti-LPA mAb-B7, every 3 days starting 1 day prior to Matrigelimplantation. Plugs were stained for endothelial CD31, followed byquantitation of the micro-vasculature formed in the plugs. Quantitationdata were means+/−SEM of at least 16 fields/section from 3 plugs. Theplugs from mice treated with the anti-LPA mAb B7 demonstrated aprominent reduction in blood vessel formation, as assayed by endothelialstaining for CD31, compared to the plugs from saline-treated mice.Quantification of stained vessels demonstrates a greater than 50%reduction in angiogenesis in Caki-1-containing plugs from animalstreated with mAb B7 compared to saline-treated animals. This was astatistically significant reduction (p<0.05 for mAb B7 vs. Saline asdetermined by Student's T-test) in tumor cell angiogenesis as a resultof anti-LPA mAb treatment.

Anti-LPA mAbs Reduces Tumor Progression in Renal and PancreaticXenografts

The anti-LPA antibodies have been shown (above) to be effective inreducing LPA-induced tumor cell proliferation, migration, protectionfrom cell death and cytokine release in multiple human tumor cell lines.mAbs B58 and B7 were next tested in a xenograft model of renal andpancreatic cancer. Below are preliminary results that demonstrate thepotential anti-tumorigenic effects of the anti-LPA antibody approach.

Tumors were developed by subcutaneous injection of Caki-1 and Panc-1human tumor cells into the left flank of 4 week old female nude (NCrNu/Nu) mice using standard protocols. After 10 days for Caki-1 and 30days for Panc-1, when solid tumors had formed (˜200 mm3), mice wererandomized into treatment groups. Treatment was initiated by i.p.administration of 25 mg/kg of the anti-LPA mAbs or vehicle (salinesolution). Antibodies were administered every three days for theduration of the study. Treatments consisted of 25 mg/kg of the anti-LPAmAb B58 for caki-1 tumors, mAb B7 for Panc-1 or Saline. Data are themean+/−SEM of 7 saline and 6 B58-treated mice for the caki-1 study and 4saline and 5 B7-treated mice for the panc-1 study. Tumor volumes weremeasured every other day using electronic calipers and the tumor volumedetermined by the formula, W²×L/2. Animals were subsequently sacrificedafter tumors reached 1500 mm³ in the saline group. Final tumor volumesand weights were recorded.

In this preliminary experiment, the ability of the anti-LPA mAbs toreduce tumor volume was apparent after the tumors reached approximately400-500 mm³. At this point, the tumors from the control animalscontinued to grow, while the tumors from the anti-LPA mAb-treatedanimals exhibited a slower rate growth in both xenograft models. Datademonstrates that the anti-LPA mAb also reduced the final tumor weightsof caki-1 and panc-1 tumors when compared to tumor weights fromsaline-treated animals.

Anti-LPA mAbs Modulate Levels of Circulating Pro-Angiogenic Cytokines inAnimals with Tumors

The anti-LPA mAbs (B58 and B7) also influenced the levels of circulatingpro-angiogenic cytokine. In animals treated with the anti-LPA mAb7(Panc-1), the serum level of interleukin-8 (IL-8) was not detectable inany antibody-treated animals, whereas IL-8 serum levels were detectablein Panc-1 and Caki-1 xenografts after 85 and 63 days, respectively. Moreimportantly there was a strong correlation (r=0.98) between tumor sizeand IL-8 levels. In the animals bearing Caki-1 tumors the serum levelsof human IL-8 were also reduced by the treatment with anti-LPA mAb58(r=0.34) when compared to saline treatment (r=0.55). As mentioned above,the reduction of circulating cytokine levels is believed to be due to adirect inhibition of cytokine release from the tumor cells themselves.These data demonstrates the ability of the anti-LPA mAb to reduce tumorprogression while also reducing the levels of circulating pro-angiogeniccompounds.

Anti-LPA mAbs Reduces Tumor Progression in a Murine Model of Metastasis

One important characteristic of tumor progression is the ability of atumor to metastasize and form secondary tumor nodules at remote sites.In vitro studies described hereinabove have demonstrated the ability ofLPA to induce tumor cells to escape contact inhibition and promotemigration in a scratch assay for cell motility. In these studies, theanti-LPA mAbs also inhibited LPA's tumor growth promoting effectors. Theefficacy of the anti-LPA mAb to inhibit tumor metastasis in vivo. Thephenomenon of tumor metastasis has been difficult to mimic in animalmodels. Many investigators utilize an “experimental” metastasis model inwhich tumor cells are directly injected into the blood stream.

Blood vessel formation is an integral process of metastasis because anincrease in the number of blood vessels means cells have to travel ashorter distance to reach circulation. It is believed that anti-LPA mAbwill inhibit in vivo tumor cell metastasis, based on the finding thatthe anti-LPA mAb can block several integral steps in the metastaticprocess.

Study: The highly metastatic murine melanoma (B16-F10) was used toexamine the therapeutic effect of three anti-LPA mAbs on metastasis invivo. This model has demonstrated to be highly sensitive to cPAinhibitors of autotaxin. 4 week old female (C57BL/6) mice received aninjection of B16-F10 murine melanoma tumor cells (100 uL of 5×10⁴cells/animal) via the tail vein. Mice (10 per group) were administered25 mg/kg of the anti-LPA mAb (either B3 or B7) or saline every threedays by i.p. injection. After 18 days, lungs were harvested andanalyzed. The pulmonary organs are the preferred metastatic site of themelanoma cells, and were therefore closely evaluated for metastaticnodules. The lungs were inflated with 10% buffered formalin via thetrachea, in order to inflate and fix simultaneously, so that even smallfoci could be detectable on histological examination. Lungs wereseparated into five lobes and tumors were categorized by dimension(large≧5 mm; medium 1-4 mm; small<1 mm) and counted under a dissectingmicroscope. Upon examination of the lungs, the number of tumors wasclearly reduced in antibody-treated animals. For animals treated withmAb B3, large tumors were reduced by 21%, medium tumors by 17% and smalltumors by 22%. Statistical analysis by student's T-test gave a p<0.05for number of small tumors in animals treated with mAb B3 vs saline.

As shown in the above examples, it has now been shown that thetumorigenic effects of LPA are extended to renal carcinoma (e.g.,Caki-1) and pancreatic carcinoma (Panc-1) cell lines. LPA induces tumorcell proliferation, migration and release of pro-angiogenic and/orpro-metastatic agents, such as VEGF and IL-8, in both cell lines. It hasnow been shown that three high-affinity and specific monoclonal anti-LPAantibodies demonstrate efficacy in a panel of in vitro cell assays andin vivo tumor models of angiogenesis and metastasis.

Example 11 Immunohistochemistry of Tumor Biopsy Material

The purpose of this example is to demonstrate that mAbs developedagainst S1P could be used to detect S1P in biopsy material. Thisimmunohistochemical (IHC) method assesses the level of S1P in the tumor(which is believed to be produced by the tumor itself) and may be moresensitive and specific than measuring protein or RNA expression ofsphingosine kinase. In addition, the IHC method would not sufferdiminution of the S1P signal as S1P secreted from the tumor is dilutedinto the extracellular space (e.g., plasma compartment). We analyzed S1Pcontent in U937 human tumor sections (frozen; 10 μm thick) from a mouseMatrigel/xenograft model. U937 cells (human lymphoma cell line; ATCC catno #CRL-1593.2) were mixed with Matrigel matrix, at a concentration of10.5 mg/ml. 600 μL of Matrigel mix containing U937 (30×10⁶ cells/plug ina 600 μl volume) were implanted into the right flank of 4-6 weeks nu/nufemale mice and allowed to grow for 30 days. The animals were sacrificedand the Matrigel plugs were excised and embedded in OTC and flash frozenin dry ice and isopentane. Then were sectioned using a cryostat to 5 umsections. Sections were then fixed in 10% neutral buffered formalin,(Sigma, St. Louis Mo.; catalog number: HT 50-1-1; lot #025K4353) for 20min at room temp and then sections. The sections were washed with 100 mMglycine (pH 7.4) in PBS for 5 min at room temp, washed 2× with PBS/0.1%Tween 20. Sections were blocked in 1% BSA/PBS/0.05% Tween for 20 min atroom temp. Primary antibodies (e.g. murine anti-S1P mAb) were diluted(1:25 or at 1:50, as indicated) in 1%/BSA/PBS/0.05% Tween and incubatedwith tumor sections for 3 hr at room temp. Sections were then washed 3×with PBS/0.1% Tween with gentle agitation. Diluted secondary antibodies(FITC-conjugated anti mouse Ab (1:250) and RRX-conjugated anti-rat Ab(1:2500 or 1:500) in 1% BSA/PBS/0.05% Tween were incubated with tumorsections for 1 hr at room temp. Sections were then washed 6× at 5 minintervals with PBS/0.05% Tween. Sections were counterstained with DAPI(4′,6-diamidino-2-phenylindole dilactate (DAPI, 10 mg; Sigma, St. LouisMo.; catalog number D3571, lot 22775) by incubation with DAPI (1:5000)diluted in PBS for 20 min at room temp. Sections were then washed 2× at5 min intervals with PBS and 1× with DI H₂O and mounted in Gelvitolmounting media and let dry. Primary antibodies used were LT1002 (LH-2;15 mg/ml) anti-S1P mAb diluted to 1.0 mg/ml and added at a workingconcentration of 1:25 in 1%/BSA/PBS/0.05% Tween. Secondary antibodiesused were: Fluorescein (FITC)-conjugated rabbit anti-mouse IgG (H+L)(Jackson ImmunoResearch, West Grove Pa.; catalog #315-095-003; lotnumber: 67031) Ab diluted 1:250 in 1%/BSA/PBS/0.05% Tween. Images werecaptured with a DeltaVision deconvolution microscope system (AppliedPrecision, Inc., Issaquah, Wash.) The system includes a Photometrics CCDmounted on a Nikon TE-200 inverted epi-fluorescence microscope. Ingeneral, 8-10 optical sections spaced by ˜0.2 um were taken. Exposuretimes were set such that the camera response was in the linear range foreach fluorophore. Lenses included 20× and 10×. The data sets weredeconvolved and analyzed using SoftWorx software (Applied Precision,Inc) on a Silicon Graphics Octane workstation.

S1P could easily be seen in tumor biopsy images using this IHC method,using the anti-S1P mAb as the primary antibody. In contrast, S1Pstaining was absent in control samples from which the primary antibodywas omitted.

Without being bound by theory or limited to these examples, it isbelieved that the measurement of the biomarker S1P could be used inconjunction with measurements of gene expression for S1P receptors andof sphingosine kinase, both of which could serve as surrogate cancermarkers. Examples of methods of gene expression analysis known in theart include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,2000, 480, 17 24; Celis, et al., FEBS Lett., 2000, 480, 2 16), SAGE(serial analysis of gene expression) (Madden, et al., Drug Discov.Today, 2000, 5, 415 425), READS (restriction enzyme amplification ofdigested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 25872), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc.Natl. Acad. Sci. U.S.A., 2000, 97, 1976 81), protein arrays andproteomics (Celis, et al., FEBS Lett., 2000, 480, 2 16; Jungblut, etal., Electrophoresis, 1999, 20, 2100 10), expressed sequence tag (EST)sequencing (Celis, et al., FEBS Lett., 2000, 480, 2 16; Larsson, et al.,J. Biotechnol., 2000, 80, 143 57), subtractive RNA fingerprinting (SuRF)(Fuchs, et al., Anal. Biochem., 2000, 286, 91 98; Larson, et al.,Cytometry, 2000, 41, 203 208), subtractive cloning, differential display(DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316 21),comparative genomic hybridization (Carulli, et al., J. Cell Biochem.Suppl., 1998, 31, 286 96), FISH (fluorescent in situ hybridization)techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895 904) andmass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000,3, 235 41).

All of the compositions and methods described and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods. All such similar substitutes and modificationsapparent to those skilled in the art are deemed to be within the spiritand scope of the invention as defined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications, including those to which priority oranother benefit is claimed, are herein incorporated by reference to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

We claim:
 1. An isolated, biologically active, murine monoclonal,chimeric, or humanized antibody or antigen-binding fragment thereof thatspecifically binds a bioactive lysophosphatidic acid to decrease theeffective concentration of such bioactive lysophosphatidic acid in vivo,wherein said antibody or antibody fragment does not bind keyhole limpethemocyanin.
 2. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of an isolated biologically active, murine monoclonal, chimeric,or humanized antibody or antigen-binding fragment thereof according toclaim
 1. 3. An isolated, biologically active, murine monoclonal,chimeric, or humanized antibody or antigen-binding fragment thereof thatspecifically binds a bioactive lysophosphatidic acid and exerts abiologic effect, wherein said antibody or antibody fragment does notbind keyhole limpet hemocyanin.
 4. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of an isolated, biological active, murine monoclonal,chimeric, or humanized antibody or antigen-binding fragment thereofaccording to claim
 3. 5. An isolated, biologically active, murinemonoclonal, chimeric, or humanized antibody or antigen-binding fragmentthereof according to claim 3 wherein the biologic effect is selectedfrom among: decreasing the effective in vivo concentration of thebioactive lysophosphatidic acid; inhibition of angiogenesis; andinhibition of inflammation.